Nikon A1Rsi Laser Scanning Confocal Microscope Information • Locaon: – TCHRF (Building R) Room 3024 • Facility is open 24/7 to trained and badged users • Book systems through hp://research.cchmc.org/mrbs – Click on “confocal” – Click on “3024 Nikon A1Rsi inverted” • Assistance on systems: – Mike’s desk is in 3006 – Ma’s office is 3442 Technical Information • Microscope: – The microscope is a Nikon Eclipse Ti inverted. It is a four-port, fully automated inverted microscope. It includes an motorized XY stage, high speed piezo Z stage stepper, and the following six objecves: • 4x Plan Apo • 10x Plan Apo λ • 20x Plan Apo VC DIC • 40x Apochromac λS DIC- Water Immersion (Note: not Planar) • 60x Plan Apo IR DIC- Water Immersion • 100x Plan Apo TIRF DIC- Oil Immersion – X and Y movement is controlled via the joysck to the right of the scope. Z movement is controlled either by the knob on the right side of the joysck or by the large focus knobs on either side of the scope. Z-speed can be changed to course, fine, or extra-fine with selectors next to the focusing knobs. – Objecves and filters should be changed via the NIS-Elements soware, do not use the buons on the microscope. • A1R Controller/Lasers: – There are four lasers on the system: • 405 • Mulline Argon (457, 471, 488, 514) • 561 • 647 – The DU4 detector has four detecon channels on photomulplier tubes that can detect in the 400-820nm range. There is also a spectral detector on this system, a total internal reflecon system, and a stochasc opcal reconstrucon microscopy system on this microscope. Technical Information • A1R Controller/Lasers (connued): – There are two types of scanners on the microscope: galvanometric and resonant. The galvanometric scans at ~1 frame per second at 512 x 512 pixels. Fast scanning is usually done with the resonant scanner, which oscillates at a connuous 7.8 kHz, and can scan up 30 frames per second at 512 x 512 pixels . Faster scanning can be accomplished with both scanners, under specific circumstances. • Max image size with the galvano scanner is 4096x4096. • Image size is limited to 512x512 with the resonant. – Confocality is achieved using the pinhole. This eliminates out-of-focus fluorescence and enables opcal seconing and eventual 3-D reconstrucon of images. The pinhole on the Nikon A1R is variable from 12 to 256μm. Opcal secon thickness is dependent on the pinhole diameter, the wavelength of light used, and the numerical aperture (NA) of the objecve. Basic Laser Confocal Microscope Theory • Terms: – Eyepiece: the poron of the microscope that you look through. These opcs commonly have a magnificaon of 10x. – Nosepiece: The poron of the microscope that one or more objecves is mounted. – Objecve: the objecve is the set of opcs that gathers light from the specimen being observed and focuses that light into a real image. – Stage: the stage is the locaon where the specimen being imaged is placed. – Laser: a device that emits coherent light arising from the smulaon of a gain medium. Lasers can be fixed- wavelength, or tuneable. – PMT: an acronym for photomulplier tube. A PMT gathers photons and mulples the electric effect of them by inducing secondary electron emission across several voltage-posive dynode stages. The mulplicave effect can be on the order of 105-107 electrons produced per single photon. – Collimator: a device that narrows and aligns non-parallel incident light. – Dichroic Mirror: meaning “two-colored,” a dichroic mirror will allow light of a certain range of wavelengths through, and reflect light of differing wavelengths. – Filter: Much like the dichroic mirror, a filter will only let a certain type of light through. They are organized into three major types: Bandpass (which transmit across a band of wavelengths), shortpass (which aenuate longer wavelength light), and longpass (which aenuate shorter wavelength light). – Fluorophore: A chemical compound that emits light upon excitaon. – Epifluorescence: A type of fluorescence produced via reflected light, as opposed to transmied light. – Autofluorescence: Natural fluorescence by certain biological structures where no fluorophore is added. – Pinhole: a variable-size iris (in our microscopes, anyway) that spaally limits incoming light. This is the mechanism that provides confocality, and eliminates out-of-focus fluorescence. Basic Laser Confocal Microscope Theory • Terms: – Resoluon: the ability of an opcal system to resolve two nearby objects separately. The higher the resoluon of the system, the smaller the distance that can be accurately imaged. There are two primary types of resoluon, and both are diffracon-limited: lateral resoluon, and axial resoluon. Lateral resoluon is in the X-Y plane and axial resoluon is in the Z plane. The resoluon of the opcal system is determined primarily by the NA of the objecve being used. An objecve with a smaller NA will have a lower resoluon, and a higher NA is higher resoluon. – Airy Disk: a diffracon paern formed by a perfect lens at its spot of best focus. it is a series of concentric circles, as it is diffracon-limited. A lens with a larger NA will have a smaller central disk, thus giving greater resolving power. – Rayleigh criterion: this is the accepted criterion for minimum resolvable detail. If the central disk (maxima) of one Airy paern intersects with the first minima of a second, then the two objects can be just resolved. If they are closer, then they are not resolved, and if they are farther apart, then they can be well resolved. – Nyquist Theorem: this establishes the minimal sampling rate necessary to be able to accurately reconstruct the object being imaged. The sampling rate is determined by the scan size and the scan area. These two parameters determine the pixel size. In order to properly sample, the sampling rate needs to be at least twice that of the original frequency. In this case, the pixel size should be at least half the size of the smallest object you want to resolve. Oversampling is acceptable, and is actually very common, but it will not add any addional informaon to the image. Basic Laser Confocal Microscope Theory – Light Path: In the Nikon A1 systems, the light follows a scan-descan path. The light leaves the lasers, enters the scan head, moves through the opcal train to the sample, and excites it. The fluorescence is then collected in the objecve and returned to the scan head. Since fluorescence is red-shied, it can be re-directed via a dichroic mirror to exit out the rear opcal output ports. This process (where the light is directed with the scan mirrors in both direcons) is known as descanning. Aer that, the light moves to the A1 controller where it interacts with the PMTs and the signal is converted to an image in the computer. Basic Laser Confocal Microscope Theory Objecve Specificaons and markings : choosing the right objecve is crical to imaging success! • Manufacturer: who made it • Magnificaon: the rao between apparent and actual size of the object being viewed. Manufacturers make objecves with mags varying from 0.5x to 250x. • Opcal Correcons: There are two primary opcal correcons made in lenses: field correcon and color correcon. • Field correcon is either planar (usually denoted as Plan), or it is curved (no correcon printed on barrel) • Color correcon comes in four flavors: monochromac, achromac (denoted as Achro or Achromat-two color corrected), fluorite (Fluor, Fl, Fluar-three color correcon), and apochromac (four colors corrected, labeled as Apo) • Numerical Aperture (NA): the light acceptance angle which in turn determines the resolving power and depth of field of the objecve • Tube length: this is the length of the body of the microscope, between the objecve and the eyepieces. Most modern objecves are corrected to infinity. • Cover glass thickness: this can either be a single number, or a range if there is a correcon collar. This is the opmal thickness of cover glass that can be used with this objecve. Basic Laser Confocal Microscope Theory • Working distance (WD): this is the distance between the front lens of the objecve and the cover glass when the specimen is in focus. Generally, higher mags = lower W, but long working distance (LWD) and extra/super/ultra-long working distance objecves (ELWD, SLWD, and ULWD, respecvely) exist. • Special properes: this will denote if this objecve can be used for certain types of contrast, like Phase (denoted Ph) or differenal interference (denoted DIC) • Immersion medium: this will be printed on the objecve barrel, or denoted by a colored ring. • Water is shown by a white ring, and W or Water is inscribed on the barrel • Oil is a black ring, and Oil or Oel is printed • Glycerol is denoted by an orange ring, and Gly is on the barrel • Special immersion media, or mul-immersion objecves are given a red ring • Magnificaon Color Codes (for objecves found on our systems): • 4X: Red • 10X: Yellow • 20X: Green • 40X: Light Blue • 60X: Cobalt Blue • 100X: White Basic Laser Confocal Microscope Theory • Aberraons and oddies: – Chromac aberraon: an aberraon where different wavelengths of light are not focused in the same plane(axial), or the different colors focus they focus at different posions in the same focal plane (transverse) This can manifest itself in several ways. Either each color will appear in a different plane, or as spots in the same plane. Objects can also appear to have colored edges (known as purple-fringing). This is corrected by placing two types of glass together, usually crown glass and fluorite., and adjusng them so that the light focuses in the same spot in the same plane. At our level, chromac aberraon can be migated by using an achromac or apochromac lens (depending on the desired number of colors corrected).