© 2011 Nature America, Inc. All rights reserved. Diffraction, Diffraction, the and pattern the PSF Airy specimen point of view, we selected the PSF method. is the measurement of the resolution of the imaging system from a containsthatimage interference no sion wavelength), an image which is completely incoherent and an (fluorescenceemis light of frequencyrelativelylower a on based contrast, imaging fluorescent microspheres results in measurements coherent,containingconstructivedestructive interference. and In length), and results in the formation of an image that is completely based on a relatively higher frequency of light (excitation laser equivalent.wave Imaging the mirror slide results in measurementsor PSFs. Although both methods measure resolution, thatthey are not are centmicrospheres, referredspreadgenerallypointfunctions toas fluores sized sub-diffraction of collected be can stack image an image the from directly measured resolution the slide with an appropriately mounted cover slip can be imaged and of an objective lens and, in effect, of the microscope itself. A mirror optical most element,important the objective lens. performance of a confocal microscope system and the quality of its sources, such as fluorescent microspheres, are ideal for assessing the lenses must exceptionallybe of quality.high Sub-resolution point must (DIC) prisms be removed from the path,light and objective contrast interference differential as such elements optical certain clean, be must components optical images, fluorescent limited collect to order In performance. ideal for optimized and maintained be must they systems, sophisticated increasingly from the most accurate image representations acquired with these as well as sciences, much of the life physical sciences.the In orderof to collect fields valid information all virtually spans (CLSM) microscopy scanning laser confocal high-resolution of use The Rationale I images require an additional 2–3 h. overnight drying period. and solutions to improve microscope performance are provided. objective lens, confocal laser scanning components and other relay optics. ofIdentification possible causes of problems with the quality of the microscope’s images. and perform fluorescent microsphere samples, set up a confocal microscope to properly collect 3D confocal image data of the microspheres T Published online 10 November 2011; doi:10.1038/nprot.2011.407 3 1 Richard W Cole ensure quality control to determine confocal microscope resolution and Measuring and interpreting point spread functions travels though the optics of the microscope, including the objective The light originating from a sub-resolution fluorescent microsphere Life Life Sciences Complex Imaging Facility, McGill University, Montreal, Quebec, Canada. Correspondence should be addressed to C.M.B. ([email protected]). Wadsworth Center, New York Health,of State Department Albany, New York, USA. NTRO his his protocol outlines a procedure for collecting and analyzing point spread functions ( There are two major methods used to determine the resolutiondeterminetothe used Theremethods aretwomajor D UCT I PS ON F F measurements. 1 , Tushare Jinadasa T he he microscope setup requires 2 h (1 h for laser warm up), whereas collecting and analyzing the T he he analysis of the 2 .paper this in purpose Asthe 2 & Claire M Brown 1 . Alternatively,.

PS diffraction- F F is used to determine the resolution of the microscope and to identify any T he he PS F F geometry is used as an indicator to identify problems with the 2 , - - - 3

T 2 he he microsphere sample preparation requires 2–3 h plus an Department of Physiology,of Department McGill University, Montreal, Quebec, Canada. by diffraction rings of many orders( of rings bydiffraction (compare object Fig. the of size actual the than larger much is that lens, and it is diffracted. The result is an image the of point source resolving power. Diffraction not only causes the light to spread spread to light the causes only not Diffraction power. resolving sample. They thereby generate the smaller PSFs and have within an increased features fine from originating light diffracted highly lens a with (NA)numerical aperture 1. of mum (FWHM)) when imaging with a ×63 oil-immersion objective toappear be maxi ~350 nm in at diameter half (at the full-width emits will source then point the which in which image an in results light, laser 530-nm 488-nm a with excited microsphere), pol labeled (fluorescently source point diameter 100-nm a example, For java/imageformation/airydiskbasics/index.html). between the diffraction rings (http://micro.magnet.fsu.edu/primer/ as in the red region, will give a larger Airy disk with greater spacing rings. Longer light,wavelengthsthe diffraction such of of spacing closer with source point sub-resolution a of image smaller a give wavelengths, such as in the blue region Shorter of the object. visible spectrum will the of size the (iii) and lens objective the of ture emitted from the fluorescent microsphere, (ii) the numerical aper rings will depend on three things: (i) the wavelength of light being diffraction the between spacing the and disk Airy the of size The PSF characteristics referredgenerally to as a PSF. from the point source is spread by diffraction, this pattern is more disk Airy the center its and pattern, Airy the coined thus was pattern resulting The astronomer. and mathematician by Biddell Sir George Airy, described first was fraction an English light waves. This spreading out of light from a point source by dif the of interference destructive the by caused are rings bright the originating from the point source, whereas the dark areas between waves light diffracted of interference constructive the from result High-NA lenses collect a larger cone of light, including the more

1a , b ). This larger central point in the image is then surrounded PS natureprotocols Fs). Fs). It describes how to prepare Fig.

| VOL.6 NO.12VOL.6 1 ). The diffraction rings rings ).diffraction The PS F F abnormalities protocol 3 . As the light light . the As | 2011 PS

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© 2011 Nature America, Inc. All rights reserved. objects from one another, when they are in close proximity.We close in are they when another, one from objects The resolution an of optical system is its toability twodistinguish Resolution above and below the in-focus image plane are visible ( ( broadershapemuch a has PSF the ( natethediffraction rings, resulting compactPSFainawith shape focus planes; therefore, small pinhole settings (e.g.,volumes. 1The majorityAiry of unit)the diffractedelimi light will come from out-of- light,thusimproving resolutionthe image contrast3Dthe and in confocalout-of-focusmicroscopy,Inblock to used is pinholethe Laser scanning confocal PSFs microscope in the the sub-resolution object is extended along the optical train of the ( direction axial ~1 be to appear would source Therefore, in the example presented above, the image the of point axis. lateral the of that times three at estimated be can axis axial (compare plane image lateral the in than pronounced more even is plane image ( also along the axial axis in the direction of the propagation of light from the hourglass shape of the PSF 3D isosurface ( (5 Airy units), the PSF is much larger and the diffraction pattern outside of the focal volume is evident compact and the diffraction rings outside of the focal volume are mostly absent. With a large pinhole ( 100-nm-diameter green microsphere collected using a ×63/1.4 NA oil-immersion objective lens. Figure 2 out along the lateral optical axis of the microscope ( of the Airy disk or the zero-order diffraction spot and the center of the first dark diffraction band within the Airy pattern. pattern showing the minimum distance that the two objects can be apart in order to resolve them by the Rayleigh criterion. The red patterns.lines indicate( the center the visualization of the dim diffraction ring contrasted with a the wavelength of 557 nm for the and lens (1.3 NA) with theoretical PSF for a oil-immersion objective and the of a 100-nm point source in the functions. ( Figure 1 1930 much higher resolution along the a Fig. 2a Fig. protocol a – 0 d yz z y ) ) With a small pinhole (1 Airy unit), the 3D isosurface ( 0.5 -axis spacing of 20 nm, with an emission

plane ( 1c | 0.8 VOL.6 NO.12VOL.6 Confocal pinhole yz 1.0 µ

, , m | | c d PSF schematics and theoretical plane ( Microsphere images and isosurfaces from a confocal microscope. Confocal images of a e 1 Airyunit ). However, at large pinhole settings (e.g., 4–5 Airy units) ). This spreading or distortion of the light in the axial axial the in light the of distortion or spreading This ). a 1.5 ) A schematic of the diffraction – d d X x [ µ 2.0 ). The images in m] ) Schematic representation c 2.5 γ Fig.1 xy ). Simulated images for a factor of 0.5 to enhance Fig. 1b Fig. -axis pixel sizes of 23 nm 3.0 | z- 2011 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 0 0.5 axis axis image plane (

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[ 2.5 µ 3.0 m] ). What this means is that the 3D image of of image 3D the ).that is means What this 3.5 , z | d

xy natureprotocols ). In fact, the size of the PSF along the the along PSF the of size the fact, In ). b 0 b plane ( xy and y z 0.5 plane ( axis when the pinhole is set to 1 Airy unit. Scale bar, 0.8 0.8 Confocal pinhol 1.0 µ µ d m were m in diameter (FWHM) in the the in (FWHM) diameter in m 5 Airyunits b 1.5 ) and a

Fig.2 X ) x [ µ 2.0 m]

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2 a Fig. 1a ). ) ) and the image ( Fig. 2 x d ). ). The confocal microscope has a , d x b ). ), but Diffractio rings Image ofthepointsource ­ y b n z c (Airy pattern) Lateral PSF ) ) of the PSF are d for the for resolution the formulae, resolution theoretical different ­several from sub-resolution fluorescentgenerated microspheres. PSF Although there 3D are the of FWHM the is planes, axial and lateral ( pattern diffraction the in band interference destructive first the to PSF the of center the resolvetwo points they must be to no closer together than the order distance from in that states which criterion, Rayleigh the use following formulae,­following which represent the Rayleigh criterion to understand and calculate. estimate the image resolution and they are relatively straigh have chosen the two expressions above, because they appropriately (e.g., emission wavelength, sample thickness and pinhole size). We taking into account many other factors that affect image resolution microscope. Some are and relativelysome are straightforward complex,highly confocal the of resolution axial and lateral the for literature the in lens.manyare expressions There objective the of index of the immersion medium and NA is the numerical aperture where x λ

confocal microscope can be calculated on the basis of the the of basis the on calculated be can microscope confocal exc x is the excitation wavelength of the laser, µ disk Air m.

y

y Axia c z Latera r l esolutio z be used for a variety of other applications. applications. other be of for used a variety also can PSFs measured and Theoretical are in surements available the literature mea experimental and calculations PSF Detailed publications regarding applications PSF theory, measurements and microsphere sample preparation. the of quality the and used being lens tive entire imaging system, including the objec also be used to determine the quality of the can PSF the of shape The microscope. the of resolution theoretical the of 10–40% within be should microsphere fluorescent sub-resolution a from PSF the of size the conditions), ideal (under lens objective high-quality a with system optical lution r l Fig.1 rm u eprec, o a high-reso a for experience, our From esolutio y = n e d ). The measured resolution, in the the in resolution, measured ).The = n (n Axial PSF 0.88 n - z 0.51 NA 2 2 l N - l ex ex y c ) A c e n Minimum distanceto is the refractive resolve twoobject

theoretical x t forward 4 ,5 : (2) (1) 6– s 8 - - ­ .

© 2011 Nature America, Inc. All rights reserved. 1| P PROCE • • • • • • • • • • • • • • EQUIPMENT • • • • • • • • • • • • • • • • REAGENTS M fluorescent microsphere samples, on guidelines how to 3D collect diodes light-emitting newer including sources, illumination a of microscope’s wide-field the quality PSF can to be used assess deconvolution image 3D accurate highly for ing allow algorithms, deconvolution image 3D for input an as used concentrations fluorophore or lipid protein, correlation absolute into convert amplitudes to function order in techniques microscopy corre with lation combination in used be can estimate volume microscope. This confocal excitation the in the beam laser estimate focused to the of used volume be can PSF the example, For  Cotton swabs Aluminum foil Latex gloves or nitrile Kimwipes (Kimberly-Clark) waterSonicating bath Forceps Bunsen burner Beakers Conical tubes, 15ml RAM) of Computer (Pentium running 4or higher Windows XP, 2GB or more with metroloj:star ( plug-in MetroloJ with (http://fiji.sc/wiki/index.php/Fiji) software Fiji Zen software (Carl Zeiss) Argon ion laser (Melles Griot; EQUIPMENT see SETUP) inverted microscope isolationairtable; Carl Zeiss) on avibration oil-immersion objective lens) attached to aZeiss motorizedAxioObserver Zeiss 710laser-scanning confocal microscope or equivalent (×63/1.4NA, Green dye, e.g., Alexa Fluor 488(Invitrogen) Fluorescent slide plastic (Chroma Technology) water (dH Distilled Ethanol that does notcontain ammonia) Lens cleaner dH diluted 1:5with Tiffen (Royal Photo) or Ross (SPI Supplies) lens tissue REAGENTsee SETUP) Labs, immersion LDF(Cargille oil,Cargille cat. type no. 16241; REAGENTsee SETUP) Fisher Scientific,Cytoseal medium (Thermo mounting cat. no. 8310-16; REAGENTsee SETUP) ProLong medium (Invitrogen, Gold mounting cat. no. P36934; Microscope slides, (Fisher Scientific, Fisherbrand cat. no. 22-178-277) 0.190 mm, Fisher Scientific, cat. no. 12-520B) Cover slips (no. 1.5, 22mm×mm, thickness between 0.160and Carl Zeiss, cat. no. 474030-9000) Cover slips (18mm×18mm, be 0.170±0.005mm, certified to SETUP) REAGENT see (Invitrogen; colors various nm, 100 Microspheres, Microspheres sampler kit(Invitrogen, cat. no. T-7284) Green-yellow microspheres, 175nm(Invitrogen, cat. no. 7220) Green-yellow microspheres, 100nm(Invitrogen, cat. no. F8803) reparation of fluorescent microsphere slides ATER

This protocol provides instructions for preparing sub

CR Vortex the bottleof microspheres. I T D I I ALS URE CAL http://imagejdocu.tudor.lu/doku.php?id=plugin:analysis: t

) STEP 9–1 2 3 O) . In addition, the PSF measurements can be be can measurements PSF the addition, In . Any microsphere sample that is used must be sub-resolution for the objective lens being tested. 2 O (e.g., GlassPlus or equivalent cleaner 14–1 ● 7 . Finally, the the .Finally,

-

T

resolution IMI

N 1 G 8

. 14h45min - -

should be no larger than 175 nm for testing high-NA immersion-objective 100 slides, although hardening mounting medium can be used. Microspheres This kit also contains a non-hardening mounting medium for making up to point source kit that includes blue, green, orange and deep red microspheres. 175-nm diameters. The 175-nm microspheres come as part of a microscope Fluorescent microspheres REAGENT SETUP ( manufacturers major the of all from microscopes laser-scanning confocal on measurements PSF making for procedures outline of detailed Finally, system. set the a of resolution theoretical the to close not is size its and/or symmetric not is PSF the of shape optical the if your system with wrong be may what troubleshoot to how on section a also is There microscopes. wide-field including system, imaging optical any to applicable is procedure the microscope, confocal 710 Zeiss a on developed was it Although images. PSF these from microscope your how of resolution on optical the measure to information and samples these of volumes image PSF the the Carl Zeiss Zen software. the of photomultiplier tube detection (PMT) channels. Collect images using dichroic mirror). Collect fluorescence emission from 500 to 600 nm one with laser onto the sample using a 488-nm main beam splitter (also referred to as a spheres the with 488-nm laser a line of 25-mW argon ion laser. Direct the Lasers, filters and mirrors EQUIPMENT SETUP Hoechst, which are excited inthe350–400-nmrange. UV range, soitisnotwellsuited blue for live-cell working with dyes, such as use at37°C. aware Be thatthe37°CHFoil may autofluorescent be inthe to aberrations Mismatching causelossesinresolution the oil andtheapplication due will are working at37°C, 37, thencat. usehigh-temperature oil (type no. 16237). a new replacement oil for LDF). room-temperature you If (23°C)work (type oils. do of offer They Labshasstopped theDFseries Cargille manufacturing Immersion oil thePSF. within or distortions aberrations cause spherical or themounting immersion medium, matchnot perfectly thatof which can medium. It noted shouldbe thattherefractive themicrospheres index of may protocol tested, shouldbe asthemicrospheres of are notsoluble types inall used, medium canbe but of the medium.slip andthemounting Othertypes from light attheinterface refraction diffracted coverhighly between theglass increasesrefraction themicroscope theresolution of by reducing thelossof andimmersion oil (1.515)well. theglass index of Having matching indices of 1.46, whereas at1.48. Cytoseal higher isslightly match These therefractive medium.mounting After curing, ProLong Goldhasarefractive index of Mounting medium smaller microspheres to make finding the 100- or 175-nm microspheres easier. cat. no. F-8813) microspheres. These large microspheres can be mixed with the microspheres sampler kit) or single-color (yellowish-green—Invitrogen, be used for low-NA lenses ( and red (580/605; cat. no. F8801). Larger 500-nm-diameter microspheres can Invitrogen: blue (350/440; cat. no. F8797), orange (540/560; cat. no. F8800) colors of 100-nm-diameter carboxylate-modified microspheres from microspheres can also be used. Recommended microspheres include various lenses. Different-colored microspheres or slides with mixed populations of Supplementary Methods Supplementary Supplementary Methods 1 9

. Labshasnotgenerated anLDF-equivalent Cargille oil for We have DF. oil, Cargille used previously type However,

We useeither ProLong Goldor Cytoseal hardening natureprotocols

< We use green-yellow microspheres of 100-nm or Excite the 100- or 175-nm yellow-green micro

0.6), such as TetraSpeck four-color (Invitrogen, ). are provided. These methods

| VOL.6 NO.12VOL.6 protocol | 2011 |

1931

-

© 2011 Nature America, Inc. All rights reserved.  view without any substantial clustering. 15| 14| to the edges of the coverslip. 13| let itfall onto the drop of mounting medium. 12| possible to the refractive index of the glass cover slip and the immersion medium (1.515).  11| spot onthe oppositeside of the coverslip. 10|  9| must travel back through the mounting medium again before being collected by the detector. of the best quality with the microspheres placed and imaged near the cover slip. In addition, the fluorescence emission light objective lenses are corrected for spherical aberration at or just below the cover slip; therefore, the PSF measurements will be the light must travel through a layer of mounting medium to reach the microspheres with this sample architecture. Most objective lens when imaging. If the microspheres are placed on the microscope slide, spherical aberrations will be high, as  8| cover slips. Therefore, no. 1.5 cover slips (or cover slips as close to 0.170 mm in thickness as possible) should be used.  the microsphere droplets tospread and create anevendistribution of microspheres onthe glass. with pointed forceps and flame them withaBunsen burner. Thiswillcreate a hydrophilic surface onthe glass, thus allowing 7| avoid any oil from fingerprints getting on glass surfaces and potentially affecting image quality.  6| microspheres canbeadded tothe suspension toaid infinding the coverslipsurface. of 70%(vol/vol)ethanol. Thiswillbethe final microsphere solution, a10 5| in an underestimate of the microscope resolution. individual microspheres for imaging. If microsphere clusters are imaged, the size of the PSF will be overestimated, resulting  aggregated microspheres and avoid the presence of microsphere clustersinthe slide preparations. 4| solution. Different dilution factors may be needed for other fluorescent microsphere sources.  second 1:100dilution for atotalstockdilution of 10 3| 2| 1932 protocol

PAUSE PAUSE CR CR CR CR CR CR Check the samplesfor the correct microsphere density—i.e., enough microspheres toget many inamicroscope field of Placethe samplesinthe dark overnight toallowthe ProLong Goldtocure. Lightly press down onthe middle of the coverslipwithacottonswabtoforce any airbubblesinthe mounting medium Pick upthe coverslipbyhand (withgloveson)orbyusing forceps and placeitata45°angle tothe vertical, and then Placea15- To aid infinding the microspheres on the microscope, apermanent marker can be usedto draw acircle around the dried

Cover the cover slips with aluminum foil in order to prevent dust from settling onto the samples. Let the solution dry for 1–2 h. Wash microscope slides with 70% (vol/vol) ethanol and wipe dry with a Kimwipe. Label the microscope slides appropriately. Vortex the final microsphere solution immediately before useand pipette 10–20 Wash no. 1.5coverslipsbyplacing them inasmall beaker filledwith70%(vol/vol) ethanol. Liftthem out of the beaker In athird 15-mlconical tube, dilute100 Place the conical tubefrom Step3(10 In asecond 15-mlconical tube, dilute100 In a15-mlconical tube, dilute100 | I I I I I I VOL.6 NO.12VOL.6 T T T T T T I I I I I I CAL CAL CAL CAL CAL CAL

PO PO

I I STEP STEP STEP STEP STEP STEP NT NT µ Store the slides at 4 °C. With ProLong Gold or Cytoseal they can last for months or longer. The solution can be left to dry for a longer period of time or even overnight. | l drop of ProLong Goldoranother appropriate mounting medium onto the microscope slide. 2011 Sonication is required, or clusters of microspheres within the sample may make it difficult to find These dilution steps are specific for the 100-nm microspheres from Invitrogen, which come as 2% solids in For optimal PSF measurements, the mounting medium must have a refractive index that is as close as The microspheres must be placed directly onto the cover slip so that they are as close as possible to the Most nondipping, high-quality immersion objective lenses are specifically corrected for 0.170-mm-thick Latex or nitrile gloves should be worn for this step and the remainder of the preparation of slides. This will | natureprotocols µ l of microsphere solution to10mlwithdH 4 stockdilution) inasonicating waterbathfor 20min.Thiswillbreak upany µ l of the sonicated 10 µ l of the 10 4 . 2 dilutedsolution from Step2to10mlwithdH 4 stockdilution to10mlwith 9mlof dH 6 stockdilution. Ifdesired, larger 0.5- 2 µ O for a1:100or10 l directly onto the coverslip. 2 dilution factor. 2 O. Thiswillbea 2 O and 900 µ m

µ

l

© 2011 Nature America, Inc. All rights reserved. of the laserlight being transmitted through the sample. The range indicator LUT canbeusedagain.Ifthe laseriswell 21| and thistestwillnot bevalid. is not properly aligned, then the transmitted light intensity image willnot berepresentative of the laseralignment ( alignment assures that both the condenser lens and the objective lens are focused at the same focal plane 20| not centered ( region of bright fluorescence should be seen in the center of the image ( center of the bright region are showing saturation, as in the example shown ( Zeiss software). This LUT is sometimes called a ‘Hi-Lo’ LUT. Set up the laser power and PMT voltage so some of the pixels at the will appear one color (red in the Zeiss software) and any pixels reading zero intensity will appear another color (blue in the software to ‘range indicator’. This will set up the image display so that any pixels reading maximum intensity (i.e.,(approximately saturated) 7–9). To help in seeing small differences in signal across the image, set the image lookup table (LUT) in the (e.g., 0.5% laser power or ~8 image the plastic slide as you would for any green dye. You will have to use a very low laser power and low detector sensitivity 19| done manually using the shape of the PSF to determine the ideal setting. needs to be adjusted accordingly. If there is no way to measure the actual thickness of the cover slip, this adjustment can be For example, if the cover slip is specified to be 0.170 mm in thickness but is slightly thinner or thicker, the correction collar  that itisproperly adjusted. 18| paper at the edges ensures that no pressure is applied directly on the front lens.  dH subsequent sweep.Repeatthisprocess twomore times: once withlens cleaner onthe lens paper( with eachsweeptoavoid transferring any dirtordust from the paperonto the lens, which coulddamage the lens during a that acleanpartof the lens papercontacts the lens atalltimes ( thumb and index finger. Sweepthe paperacross the lens three times, moving the papersideways aftereachsweepensuring a piece of lens paperand folding itthree times into along rectangle ( 17|  the lasertowarmupfor atleast1h. 16| Microscope setup showing saturated pixels in red. distribution in these images is emphasized by with a well-aligned laser ( alignment ( the microscope condenser is not in Köhler light images showing the intensity image when is aligned ( fluorescence image generated when the pinhole uniform fluorescence plastic slide, showing the ( solution and then a third piece containing dH a fresh piece of lens tissue containing cleaning remove excess oil ( and move it gently across the lens surface to paper along its long axis ( immersion objective lenses, fold a piece of lens and laser alignment. ( Figure 3 d ,

e CR CR CR 2 ) Fluorescence images generated using a O toremove any residue from the lens cleaner. Setupfor imaging using the transmitted light detector. Imaging withthe transmitted light detector generates animage Perform Köhleralignment of the transmitted light condenser inorder toverifythatthe laseriswellaligned. Köhler Ifthe confocal pinhole is useradjustable, then useagreen fluorescent plastic slide toalign it.Startbysetting upto Ifthe lens hasacorrection collar(e.g., correction for immersion medium, temperature, coverslip thickness), ensure Cleanyourobjectivelens. Thiscanbedone whilethe laseriswarming up.Remove any excess oilonthe lens bytaking Turn onthe microscope and allow I I I T T T

| I I I Lens cleaning protocol and pinhole CAL CAL CAL d f ) and when it is in Köhler alignment ) or misaligned (

STEP STEP STEP Fig. 3 b ). ( a If there are problems with the geometry of the PSF, it could be a result of the correction collar adjustment. If the objective lens is not clean there are likely to be distortions in the measured PSF. Holding the lens Ensure that all DIC optical elements are removed from the confocal light path. ● , c b ) Repeat the process with

) When cleaning e T g a ), then adjust the pinhole. If it is difficult to center the bright region, the laser may be misaligned. IMI ). ( ), hold it at the edges e ). ( d N – g G f ) The intensity , 1 h 45 min g ) Transmitted µ W and PMT gain of 500 V). Use a low zoom setting (approximately 1–2) and a fast scan speed 2 O. b a c Fig. 3 Fig. 3 Fig. 3 b ). It isimportant touseafresh area of the paper d f a Fig. 3 ). Hold the paperatthe edges betweenyour d ). If the maximum brightness in the image is d ). If the pinhole is well aligned, a circular natureprotocols Fig. 3 e g

| VOL.6 NO.12VOL.6 c ) and once with 2 0 . If the condenser protocol | 2011 Fig. 3 |

1933 f )

© 2011 Nature America, Inc. All rights reserved. Similarly tothe blacklevelsettings, ifthe laserpowerordetector sensitivity is set toohigh then the fluorescence signal can Do not usethe fast scanning mode, asnoise within the images may make itdifficulttoproperly adjustthe settings. power of ~0.5%(~8 maximum image intensity (~3,000intensity unitsina12-bitimage). Choose the 488-nmlaserline and startwithalaser 33| low-intensity data clipping isavoided (compare within the image reads zero intensity units( platform-specific information). It isbesttosetthe offset using a range indicator–type LUTto make sure that no pixel to zero. Thissetting isoften calledadigital offset, blacklevelorbackground level(see the software thataPMTintensity readout belowacertaindigital threshold isnoise and itsetsany signal belowthisvalue 32| to the 31|  30| scan lines along the ning must be used, the scanning must be carefully calibrated in order to avoid problems with the registration of adjacent  value canbefine tuned laterinthe protocol. to reduce pixel noise. Startwithazoomfactor of 2–3inorder toachieve anappropriate pixel size;however, note thatthis 5–25 29| or Leica SP5,seethe filter (barrier filter)collecting light from ~500to600nm.Ifyou are using a Zeiss710, Zeiss510,OlympusFV1000,Nikon A1 consist of the 488-nmlaserline from anargon ion laser, a488-nmmain beamsplitter(dichroic mirror) and an emission 28| A1R; Leica SP5) in the shots are provided for five major laser scanning microscope platforms (Zeiss LSM710; Zeiss LSM510; Olympus FV1000; Nikon  then asampleof 0.5 27| Instrument setup objective lens. Placeitfacing upfor anupright microscope stand and down for aninvertedmicroscope stand. 26| 25| 24| 23| 70% (vol/vol)ethanol solution. 22| your microscope service technician todo thisfor you. know how toalign the laser, then adjustthe alignment tocenter the bright region of excitation. Ifyoudo not, then call light detector ( aligned youwillseeabright region centered inthe image ( 1934 saturate the detector, thus clipping the high-intensity data (compare protocol

CR CR CR Adjust the detector (PMT)gainand laserpowersothatthe average microsphere intensity isapproximately 75%of the Setthe detector offset. Most confocal manufactures haveasoftware setting for the detector offset. Thisfunction tells Setthe PMTdetector gain.Thissetting willvaryamong manufacturers, buttypically avalueof 600–750Visideal. Refer Zoom inonthe microspheres atthe center of the field of view for the bestPSFcharacterization. Setthe image acquisition toscananimage frame of 1,024×pixels atamoderate scanspeed(pixel dwell time of Setupthe confocal light pathfor imaging agreen dye (e.g., Alexa Fluor 488/GFP).Forexample, the light pathcould Focusonthe 100-or175-nmmicrosphere sample. Ifyouneed tomeasure aPSFfor alower-resolution lens ( Placethe 100-or175-nmgreen microsphere sampleonthe microscope stage withthe coverslipside facing toward the Ifthe lens isanimmersion lens, placeadrop of oil,waterorother appropriate immersion medium onthe lens. Move the objectivelens tobetestedinto position onthe microscope. Ensure thatthe microscope focus isstableand thatthe systemisnot inanarea prone totemperature shifts. Cleanthe microscope slide withlens cleaner toremove any buffersolution and cleanany oilfrom the sampleusing a

| I I I VOL.6 NO.12VOL.6 µ T T T S s perpixel). Foroptimal intensity information itisbesttocollect12-bitimages. Line orframe averaging canbeused I I I upplementary Methods CAL CAL CAL

STEP STEP STEP Fig. 3 | 2011 Artifacts in the PSF shape can be observed when imaging at the periphery of the field of view. Set the instrument for unidirectional scanning, not bidirectional or raster scanning. If bidirectional scan This is the general instrument setup and microsphere imaging procedure. Detailed procedures with screen ● µ y

f µ S axis, which can lead to image artifacts. T W). Use the continuous scanning mode toensure thatthere are no saturated pixels withinthe image. ), verifythatthe transmitted light condenser isaligned. Ifso,then the laserismisaligned. Ifyou | upplementary Methods IMI m microspheres should be used. S natureprotocols upplementary upplementary Methods N G 30 min for more information onspecificconfocal platforms. Fig. 4a for platform-specific details helpful for performing Steps29–47. . Fig. 4a , b ). Thisensures thataccurate intensity information iscollectedand , b with Fig. 3 Fig. 4c g ). Ifthe bright region isnot centered onthe transmitted Fig. 4a , d ). , b with Fig. 4e S upplementary Methods , f ). These data points show up

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0.6 NA),

- -

© 2011 Nature America, Inc. All rights reserved. lens. Use the zoom and pixel number features of the software to make sure that the image pixel size is approximately at the Where These valuescanalsobecalculateddirectly from the lateral (equation (3))and axial (equation (4))resolution equations. the Nyquistsampling frequency needs tobe~3times smaller thanthe resolution of the objectivelens. Thisisagood approximation of the Nyquistlimitor 39| Microsphere imaging 38| biological samplesmay berequired. and interpreting the PSF; therefore, lowerdetector gainand higher laserpowersettings thantypically usedfor imaging 37| 36| your confocal microscope’s manufacturer to determine the relationship between the software’s35| pixel size and the Airy unit. be maintained across allexperiments. tings are tobeused, then they should gain to1(i.e., off). Ifother gainset a digital gainfeature. Setthe digital 34| until the saturation disappears. the image, then reduce the PMTgain there are stillsaturated pixels within minimum laserpowerisreached and until the red pixels disappear. Ifthe showing up,reduce the laserpower ( as red pixels inthe range indicator LUT intensity profile. Scale bar, 0.5 clipping, the bead shape is misrepresented in the in red. ( ( the sample is too high, the detector saturates. When the intensity of light coming from intensity data clipping in the intensity profile ( reading zero intensity ( the image shows blue pixels representing pixels ( well-adjusted image acquisition settings. profile along a line across the microsphere with fluorescence microsphere and the intensity data clipping. ( Figure 4 recommended value, but no larger. This will ensure that the sampling is high enough in order to determine the PSF accurately e c Fig. 4 , ) Saturated pixels within the image are shown d ) When the offset value is set too high, Set the pinhole to 1 Airy unit. If your confocal software does not express the pinhole in Airy units, then you should contact Collectimages withthe proper sampling frequencies in Once the instrument settings are optimized, take animage of the microspheres. Verify the image acquisition settings using the range indicator LUT. Ahigh signal-to-noise ratio ishelpful for visualizing Scananimage of the microspheres. Most confocal microscopes have f λ

e ) With both high- and low-intensity data | exc Image acquisition settings to avoid ). Ifthere are saturated pixels is the excitation wavelength, a , b ) Confocal image showing a c ), thereby causing low- ●

µ T m. IMI N 2 G 1

. The largest acceptablepixel sizesfor accurately determining the PSFare shown in 1 h 30 min

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n Ax is the refractive index of the immersion medium and NA is the NA of the objective ia La c a e s l tera amplin s l ampling gf equenc frequency x , y y and = 3 x ( n n . Inorder toaccurately measure the PSF, the pixel size = 8 0 − − f d b 1,000 2,000 1,000 2,000 . 5 0 0 0 0 0 0 . 8 3 ⋅ 2 2 1 3,00 4,00 1,00 2,00 Intensity NA l l 0 0 0 0 0 ex 0 ex 0.2 .2 NA c c 0. 0 2 ) 0. 0. 0 4 0 4 natureprotocols .4 .6 .6 0. 0 6 0. 0. 1 8 1 8 Distance Distance .8 Distance 1. ( µ .0 ( ( .0 0 µ µ m) m) m)

| VOL.6 NO.12VOL.6 1. 1. 1. 1 2 1 2 1 2 .4 protocol .4 .4 1. 1 6 | 2011 1. 1. 1 6 1 6 T able .8 |

.8 1935 .8 (4) (3) 1 .

© 2011 Nature America, Inc. All rights reserved. the  42| 41| ? not anaccurate representation of the measured PSF(compare confocal software packages often interpolate pixel valuestosmooth outthe data, thereby producing a‘pretty’ picture thatis broadening becauseof the sizeof the aggregate and the microscope resolution willbeunderestimated. Note thatthe (and soon)brighter than the dimmest microspheres. Ifthese aggregates are usedtomeasure the PSF, there couldbesome correspond toaggregates of microspheres thatare stillsub-resolution, butshould beroughly double, tripleorfour times If the microsphere sampleiswellprepared then there should bemany microspheres of similar intensity. Brighter spots 40| information about the PSF (compare bleach, image collection can take a long time and image data sets can be large, without any significant gain in resolution or and 170 nm) and the axial (  plane and 100 nm in the If If the sampling frequency is too low, the shape of the PSF cannot be determined accurately along the lateral ( for the objective lens being tested. The sampling we typically use for a ×63/1.4 NA or ×100/1.4 NA lens is 50 nm in the Note: Calculations for axial resolution were basedonthe following: T 1936 a 1.40 1.35 1.30 1.25 1.20 1.15 1.10 1.05 1.00 0.95 0.90 0.85 0.80 0.75 0.70 0.65 0.60 O protocol

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b bjective CR CR le Setupthe Crop, zoomorusearegion of interest tochoose a single microsphere ( Selectthe dimmest microspheres withinthe image toanalyze, as these are more likely to be individual microspheres. z

-stack images. a a c c c c b b b b b a a a a a a | I I VOL.6 NO.12VOL.6 1 T T I I B | CAL CAL LES

Resolution Resolution and maximum PSF sampling size. NA

H STEP STEP OOT z -stack acquisition withinthe software. | It is important to collect the microsphere images according to the proper sampling frequencies in 2011 Use the values in I N L G ateral resolution (nm)for 488-nm excitation light | natureprotocols z plane. Fig. Fig. 5 178 184 191 199 207 216 226 237 249 262 277 293 311 332 356 383 415 b , , 250 and 500 nm) directions. If the sampling frequency is too high the microspheres can T Fig. Fig. 5 able a Air ( 1 a n orcalculations basedonequation (4) to setthe spacing (i.e., interval) between with =1.0),

b Fig. Fig. 5 Water ( 488-nm excitation light n =1.33), A b xial resolution for ). c Oil (1.51)immersion medium. Fig. 5b 1,074 1,268 1,502 1,789 2,147 737 836 948 624 761 907 455 515 579 648 568 649 , d ).

Fig. 6a , b ). sampling (nm) L ateral 104 111 119 128 138 75 79 83 87 92 98 59 61 64 66 69 72 xy

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xy

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© 2011 Nature America, Inc. All rights reserved. saving the files.  extension). scan speed, laserpower)and alsosaveitasasingle 12-or16-bitmulti-TIFF fileoraseries of single TIFFfiles(.tiffile 47| from the 1Airyunitsetting are usedtodetermine the microscope’s resolution. microsphere aggregate. Arainbow LUTisusedtoseethe dimdiffraction bands withinthe PSFimage ( microspheres and a×63/1.4 NAobjectivelens. One microsphere ismuch brighter thanthe other, and soitislikely tobea microsphere PSForthogonal view inwhich the confocal pinhole issetto1Airyunit.These data were collectedwith100-nm been captured ( 46| 45| (B) ( (option B). 44| minimum stacksizeshould go to2 function isimaged, twotimes the PSFheight is best. Forexample, ifthe PSFis2 below the plane of focus. Ideally, toensure thatthe entire the 43| the smoothed data in the Zeiss 710 Zen software. Compare the images of the actual data in microspheres look blurred in the orthogonal view. Images were taken from confocal software platforms will interpolate under-sampled data, making were not set correctly, resulting in incomplete imaging of the PSF. ( ( (100 nm), oversampling (50 nm) and under-sampling (250 and 500 nm). under-sampling (80 and 170 nm). ( image plane showing ideal sampling (40 nm), over-sampling (30 nm) and a ×63/1.4 NA oil-immersion lens. ( 100-nm yellowish-green microsphere taken on a confocal microscope with Figure 5 c A ) Image of a microsphere in the a

(ii) Enter the totalnumber of slices tobeimaged orthe distance toimage aboveand belowthe center image plane. (ii) Focusabovethe microsphere of interest and mark the lastplane when youseeno intensity inthe image. ) First/last CR (i) Focusonthe center of the microspheres and click onthe center button. (i) Focusbelowthe microsphere of interest and mark the firstplane when yousee no intensity inthe image. Savethe data inthe microscope manufacturer’s proprietary format tomaintain the metadata (e.g., pixel size, Use the orthogonal viewer tolookatyourmicrosphere PSFdata inorder toensure thatthe image of the entire PSFhas Perform the Use the fast orcontinuous scanning mode and setthe Setthe z C -axis limitstoatleastthe height of the PSFaboveand I enter T

| I Over- and under-sampling when measuring the PSF. Images of a CAL

z STEP -axis limits. As ageneral rule, itisgood toset Fig. 5 z d Make sure the TIFF format is 12-bit or 16-bit and that there is no compression of the image data when -stack acquisition. . Scale bar, 0.5 b ) and thatthe xz a b ) Images of the microsphere in the plane in which the z-stack parameters ) Images in the µ m. µ m abovethe in-focus plane and 2 xz or xz b plane showing ideal yz image does not show a partial function ( d ) Many b with xy

z -stack options inone of twoways:first/last(option A)orcenter the 710 using the Zen software and settings representative of those found in Scale bar in lens. ( Zoom 3 of 100-nm microspheres taken with a ×100/1.4 NA oil-immersion Figure 6 of-focus light ( range may have to be larger because there will be more out-  Airy units. 49| 48| b a

CR S Repeatthe procedure withthe pinhole setting at4–5 Collectdata for atleastfiveindividual microspheres. upplementary upplementary Methods 50 nm b I 30 nm ) ) Image at Zoom 3 of a single microsphere enlarged for visualization. T µ

I | m belowthe in-focus plane. Using region tools to image single microspheres. ( CAL a , , 10

STEP Axial 100 nm µ Fig. Fig. 7 m; m; scale bar in z 40 nm µ Lateral pixelsize(nm axisspacing(nm) With a larger pinhole setting, the m high along the natureprotocols b ). ). The laser power may also have to Fig. 5 . 250 nm 80 nm b c , , 0.5 ). ) Figure 7 µ 500 nm m. m. Images were taken on a Zeiss z

axis, then the | Fig. 7 170 nm VOL.6 NO.12VOL.6 a shows asample d b ). The PSFdata protocol 250 nm a c | ) ) Image at

2011 z

-stack -stack 50 nm 500 nm |

1937 © 2011 Nature America, Inc. All rights reserved. sphere ( 51| averaged for five or more microsphere image stacks. each one has to be cropped from the others into a separate image stack for analysis. Report data can then be manually  microsphere data and determine the sizeof the PSF. http:// at found be can it using for imagejdocu.tudor.lu/doku.php?id=plugin:analysis:metroloj:start. manual detailed a Chooseand ‘Plugin-MetroloJ-Generate PSFreport’ to analyze the plug-in, MetroloJ The http://fiji.sc/wiki/index.php/Fiji. at maintained and updated regularly. It canbedownloaded plug-in toanalyze the PSFdata. Fijiisfreely availableand is 50| Data analysis clipping or image saturation is occurring. power and detector gain in order to ensure that no data range indicator LUT to readjust the offset (black level), laser reach the detector. Before collecting the PSF images, use the be adjusted because with the larger pinhole more light will is apparent. Scale bars, 1 image is not as distinct, but the elongation of the PSF in the the into two distinct spots, resulting in a double image of each microsphere in ×20/0.8 NA, air objective lens shows how the DIC prism splits the laser beam microspheres within the image volume. ( sub-resolution microspheres because it has a higher intensity than the other down in the objective lens. The microsphere in and ( (top) and the orthogonal views showing the Figure 7 1938 protocol

CR xy c a Review the PSFreport generated byMetroloJ. The report shows the lateral and axial views of the image of the micro Openthe b

xz xy image plane and distortions along the | ) 5 Airy units. Images were taken with a ×63/1.4 NA, oil-immersion I VOL.6 NO.12VOL.6 T

| I x Fig. 8 Ideal and distorted PSF measurements. ( -profile &fittingparameters: CAL

Intensity (AU) xy yz plane and to the right is likely to be a spot containing two 100 150 1

50 image plane (right) for a pinhole setting of ( STEP µ a m z ● 0 -stack data filesinFijiand usethe MetroloJ ). Asummary tableshows the theoretical resolution of the lens and the resolution calculatedalong the

| T 2011 The MetroloJ plug-in can only analyze one microsphere at a time, so if multiple microspheres are imaged, IMI yz µ m in N |

G natureprotocols xy 1 20 min a image plane (lower left), the , a b and and ( µ m) c b d ) Image of a PSF imaged with a that is about one-third of the way ; 5 2 z µ Line: Fitted Dots: Measured axis. ( m in Resolution table: Pinhole: 1.0Airyunits Sampling rate:0.48 NA: 1.4 Wavelength: 488.0nm Microscope: confocal Microscope infos: b a c , d b . ) At ×63, the double ) Representative PSF 3 xy a xz ) 1 Airy unit image plane image plane d c b a Parameters: R Standard deviation:2.1948 Sum ofresidualssquared:351.6443 Number ofrestarts:2(2) Number ofiterations:449(8,000) Fitted on z y x × =2.0163 =0.1203 =154.5538 =9.3571 2 0.048 : 0.9958 ×

0.194 y

= a 0.668 0.241 0.247 FWHM + µ m ( b – µ µ µ a m m m )*exp(–( c a × 20/0.8 NA—DICprisminplace Theoretical x resolution 0.349 0.139 0.139 - c ) 2 /(2* Pinhole 1Airyunit µ µ µ d m m m 2 )) (the the the fit to the data and the fitting parameters for program generates an intensity profile showing objective lens in x the measured FWHM from the fit to the PSF in imaging parameters entered into the software, of the microsphere ( the MetroloJ software generates a report that shows Figure 8 ? the also included inthe report (data along curve fitting and the fitting statics are along the through the center of the microsphere Plots of the intensity data from aline the data curvefitting (FWHM; parameters and the laterisbasedon on the basisof the image collection and , ,

y TROU and x xy x , , x z profile curve and fit is shown in , , axisare shown in y axes. The former iscalculated and xz z

| B and and the theoretical resolution of the MetroloJ report summary. ( LES z x resolution determinations , b yz H y images through the center and x OOT , , y a and ); ); the summary of the I z N axes withthe Pinhole 5Airyunits d z G ( × 63/1.4 NA—DICprism b ). ). Finally, the Fig. 8 in place c a Fig. 8 ). c – ). c

) ) The

x , - y b

). © 2011 Nature America, Inc. All rights reserved. Steps 50–53,Data analysis: 20min Steps 43–49,Imaging: 60 min Steps 39–42,Imaging: 30 min Steps 27–38,Instrument setup:30min Steps 22–26,Microscope setup: 15min Steps 19–21,Microscope setup: 30min(may belonger depending onpinhole adjustment) Steps 16–18,Microscope setup:1h(lasersshould bewarmed upfor atleast1h) Steps 11–15,Slide preparation: 12h Steps 6–10,Slide preparation: 2h Steps 1–5,Slide preparation: 45min ● T Troubleshooting advice canbefound in ? Gaussian function using the built-inImageJ curvefitting algorithm and the FWHMis determined. Data along astraight line though thisdata point isextracted along the describes the data analysis indetail. Essentially, the maximum intensity pixel withinthe entire 3Ddata setisdetermined. 53| and (2). This is done by multiplying the theoretical resolution from the report by a factor of 1.25. given by the program is very high. It is preferable to compare the resolution with the values calculated from equations (1)  52| a 51 40 S

TROU tep

b T CR le IMI Ifdesired, analyze the PSFinanother program orwithcustomsoftware. The MetroloJ websiteincludes amanual that Check the resolution of yourmicroscope withthe objectivelens being tested. I 2 T N I B | CAL G LES

Troubleshooting table. Asymmetric flare on the PSF Odd shaped or distorted PSF Blurry PSF along the PSF is elongated or compressed along the PSF is elongated or compressed dim spots of similar brightness variable and there are not many Microsphere intensities are highly P roblem

H STEP OOT The MetroloJ plug-in uses a more rigorous equation to define resolution. Thus, the theoretical resolution I z z N axis axis G T able Spherical aberration with galvanometer mirrors defective relay optic; problem Pinhole is misaligned; vibrations lens, dirty optical elements, DIC optics in place, dirty Focus drift Temperature drift Microsphere aggregation P ossible reason 2 . Additional troubleshooting guidance canbefound inreference

minute or two in a tabletop centrifuge to precipitate out period of time; spin down the microsphere sample for a Prepare new microsphere samples; sonicate for a longer S mounting medium make sure you used the proper cover slips, try different make sure there are no bubbles in the immersion oil; Clean the objective lens and apply new immersion oil; bidirectional scan Replace defective optic; try different scan speeds and/or Check pinhole by opening it up, align if necessary. same microsphere over time at the same in the light path; check for vibrations by imaging the path; clean the objective lens; clean the optical elements Make sure DIC optical elements are removed from the light for a service call for the microscope temperature drift. If the problem is still not resolved, ask a single plane over time. If not, try the solution above for Test that the focus of the microscope is stable by imaging use temperature-controlled chambers Isolate the microscope from temperature shifts: cover it, aggregates before preparing new slides xy olution , xz and yz axes. These curvesare then fittedtoa natureprotocols

| VOL.6 NO.12VOL.6 z plane protocol 8. | 2011

|

1939 © 2011 Nature America, Inc. All rights reserved. for a ×63/1.4 NA lens. T the FWHMfitvaluesalong the and circular along the optical axisthe PSFisverysymmetric, indicating agood-quality lens. The point source images should alsobeverysymmetric ProLong Goldmounting medium. These distortions inthe PSFwillworsenwhen imaging deeper into the sample likely tobecausedbyspherical aberrations due tothe slight index of refraction mismatch betweenthe immersion oiland the microspheres (right side orupperpartof the orthogonal view images), the diffraction patternisnot asapparent. Thisis The example in tion patternaboveand belowthe focal plane) and oneither side of the central optical axisinthe and the samplepreparation. The main indicator of agood-quality lens isasymmetric PSFbothalong the performing closetothe resolution limit. are representative of a×63/1.4NAoil-immersion lens the theoretical resolution. The PSFdata shown in and objectivelens being tested. Thisvalueiscompared with microscope’s resolution for the specificwavelength of light determined from the PSF, isadirect measure of the With the confocal pinhole setto1Airyunit,the FWHM, ANT green microspheres and a ×63/1.4 NA oil-immersion lens. Scale bars, 1 generated with a Zeiss Pascal 5 confocal microscope using 100-nm yellowish- is very similar to the variability seen between experiments. Images were setup and objective lens ( microspheres taken on different days, using the same imaging acquisition microspheres on the same data from the same slide ( sub-resolution 175-nm yellow-green microspheres taken of different Figure 9 1940 a Date S.d. (%) S.d. Average 09/05/2011 07/05/2011 06/05/2011 30/04/2011 27/04/2011 25/04/2011 24/04/2011 23/04/2011 22/04/2011 21/04/2011 05/02/2011 protocol The PSFmeasurements with the confocal pinhole setto5Airyunitsare meant totestthe quality of the objectivelens b le I

C | VOL.6 NO.12VOL.6 3 I

PATE | | PSF variability. (

Variation over time of the average resolution measured D RESULTS Figure 7 | 2011 0.24075 0.23675 0.0103 0.2307 0.2306 0.2386 0.2256 0.2274 0.2366 0.2034 0.2326 0.236 0.229 b ). The variability seen within an experiment a 4 x , x | b

and natureprotocols ) Representative b shows the appropriate diffraction patterns belowthe microspheres near the coverslip.Abovethe y axes. Thissymmetry canbevisualized inthe x and 0.0093 0.2441 0.2612 0.2496 0.2404 0.2404 0.2414 0.2285 0.241 0.244 0.242 0.259 0.238 y xy 4 y axes inthe MetroloJ PSFreports. , xz a and ) and from different yz images of 0.69125 0.69575 Figure 7 0.6476 0.4886 0.0661 0.5949 0.5955 0.6204 0.6086 0.5276 0.5652 0.543 0.56

11 z

µ m. a

z 4–6% for the the same session typically show astandard deviation of PSF measurements of different microspheres imaged within but should bewithin30–40%of the theoretical resolution. lenses, alwaystesttheir quality using PSFmeasurements. is not defective ordamaged. When receiving new objective turer and havethe objective lens inspected tomake sure it show similar abnormalities, contact the microscope manufac system calibration oralignment. Ifthe second lens does not ties, there couldbeanissuewiththe confocal scanning similar quality. Ifasecond lens hassimilarPSFabnormali repeating the measurement withanother objectivelens of after sequentially repeating Steps17–22,werecommend and repeat the PSFmeasurements. IfapoorPSF persists step atatime (e.g., realign the pinhole) inthe procedure To isolatethe cause of the PSFdistortions, repeat onlyone the light pathfor high-resolution microscopy applications. tion. Therefore, DIC optics should alwaysberemoved from result inasevere underestimate of the instrument resolu in adouble image of the microspheres ( light into twowaves, ordinary and extraordinary, resulting the microscope. When the DIC prismisinplace, itshears the the microscope slide, coversliporwithinthe optical pathof objective lenses; misaligned confocal pinholes; and dirton PSF data are DIC optics inthe light path( back toSteps17–22.The most common reasons for poor axisFWHM.For day-to-day measurements, the average PSF b a In general, PSFmeasurements willvaryfrom lens tolens, If the PSFdata do not show symmetric functions, then go xz xy 1 µ m 22 April Bead 1 xy yz image and verified numerically bycomparing x and y axisFWHMvalues and ~12%for the xz xy 1 µ 23 April Bead 2 m yz x and Fig. 7c y z xy xz planes ( axis(diffrac Fig. 7c 1 , 8 µ 27Apri d . Along the Bead m ); thiswill , d Fig. 7 ); dirty yz 3 l - b - ). -

- © 2011 Nature America, Inc. All rights reserved. 6. 5. 4. 3. 2. 1. com/reprints/index.htm at online available is information permissions and Reprints Published online at interests. C analyzed them and assimilated the data for the time-dependentwith critical studies. reading and editing provided by R.W.C.and T.J.interpreted collected data PSFresults. images, C.M.B. wrote the manuscriptlens quality.and designed C.M.B. the andfigures R.W.C. prepared samples,testing collected confocal images, microscope analyzed resolution, images confocal microscopeAUT quality and objective K. Young for critical reading of the manuscript. Methods for software for for thank B. Northan from Media Cybernetics for generating the theoretical PSF images help with sample preparation, PSF image stack acquisition and data analysis. We Complex Imaging Facility for portions of the work presented. We thank R. Stack for Light Microscopy & Image Analysis Core Facility, as well as the McGill Life Sciences A is availableNote: information the via HTML of version Supplementary this article. quality control metric. Further suggestions for possibleremedies topoorPSFquality are found inanarticle byGoodwin objective lenses are damaged, the PSFmeasurements should not varysubstantially. Thismakes the measurement ideal asa T FWHM valuesshow similarstandard deviations (4%for the o able cknowle

m Figure 3 Figure 1 H pet light microscopy.light three-dimensionalin used lens objective oil-immersion an in aberration 1997). Zeiss, (Carl Soc. Philosoph. Cambridge the of Trans. (2007). actions. corrective and analysis,methods, 1995). Press, Gibson, S.F. & Lanni, F. Experimental test of an analytical model of model analytical an of test ExperimentalF. Lanni, & S.F. Gibson, Beyer,H. in H. Heinz, & M. Gluch, B.,Gröbler, S., Wilhelm, aperture. circular with object-glass an of diffraction the On G.B.Airy, microspheres:fluorescent using aberration optical P.C.Evaluating Goodwin, Pawley,J.B. OR i 3

on Nikon and Olympus confocals, respectively. We thank B. Eason and CONTR n ). Therefore, unlessthe samplequality degrades overtime, samplepreparations change, equipment problems ariseor g dgm , as well as F. Waharte and M. Thibault for providing , C. Glowinski for preparing 3D PSF figures using the Bitplane Imaris

Figure 2 fi Handbuch der MikroskopieHandbuchder nanc IB ents UT Handbook of Biological Confocal Microscopy Confocal Biological of Handbook http://www.natureprotocols.com I i , A. Spurmanis for providing objective lens cleaning images

ONS al al We acknowledge the use of the Wadsworth Center’s Advanced J. Opt. Soc. Am. A Am. Soc. Opt. J. l . i nterests nterests R.W.C. and C.M.B. conceived of the need for a protocol for The authors declare no competing financial declare no The financial authors competing 2nd ed. (VEB Verlag(VEBTechnik,ed.2nd 1985).

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5 , 283–291 (1835). 283–291 , Methods Cell Biol. Cell Methods / .

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2nd ed. (Plenum ed. 2nd http://www.nature. S upplementary

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x , y axisFWHMand 11%for the 9. 8. 7. 13. 12. 11. 10. 21. 20. 19. 18. 17. 16. 15. 14.

commercial laser scanning confocal microscope.confocal scanning laser commercial a concentrationswith and dynamics protein fast measuring for (Elsevier,2007). 397–413 D.E.) Wolf, microscopy.epifluorescence in behavior confocal Partial system. microscope light a of properties imaging Sci. microscopy.correlation image by migration cell duringdynamics (2005). L33–L36 structure.time hidden the exploiting microscope: scanning microscope.scanning laser spectroscopy.correlation by disassembly and assembly adhesion during measureddynamics Paxillin (2008). 46 Soc. Microscopical Royal J. 2011). ( C.D.) Geddes, (ed. in (RICS). spectroscopy correlation image raster and microscopy live-cell for conditions Microanal. sources.conventional than microscopy biological for sources deconvolution. and deconvolution. deconvolution. (2001). passim 1082 microscopy.light in deconvolution Brown, C.M. Brown, P.C.in Goodwin, three-dimensionalof Determination D.A. Agard, J.W.& Sedat,Y., Hiraoka, Wiseman, P.W.Wiseman, M.A. Digman, M.A. Digman, E. W.W.Gratton, Mantulin, & A.R., Horwitz, C.M., Brown, M.A., Digman, Nyquist, H. Certain topics in telegraph transmission theory.transmission telegraph in topics Certain H. Nyquist, photomicrographicalpurposes. for illumination of method New Koehler,A. Optimal C.M. Brown, & A. Spurmanis, D., Zuo, C., Vining, J., Lacoste, illumination better are diodesLight-emitting Turner, J.N. R.W.& Cole, microscopy wide-field using imaging cell Live M. Platani, & J.R. Swedlow, image and microscopy fluorescence Quantitative J.R. Swedlow, image and microscopy fluorescence Quantitative J.R. Swedlow, to guide workingperson’s A J.R. Swedlow, & L.H. W.,Schaefer,Wallace, , 617–644 (1928). 617–644 ,

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