Probing Small Distances in Live Cell Imaging

hv photonics

Review Probing Small Distances in Live Cell Imaging

Verena Richter 1, Peter Lanzerstorfer 2 , Julian Weghuber 2,3 and Herbert Schneckenburger 1,*

1 Institute of Applied Research, Aalen University, Beethovenstr. 1, 73430 Aalen, Germany; [email protected] 2 School of Engineering, University of Applied Sciences Upper Austria, 4600 Wels, Austria; [email protected] (P.L.); [email protected] (J.W.) 3 Austrian Competence Center for Feed and Food Quality, Safety and Innovation, 4600 Wels, Austria * Correspondence: [email protected]

Abstract: For probing small distances in living cells, methods of super-resolution microscopy and molecular sensing are reported. A main requirement is low light exposure to maintain cell viability and to avoid photobleaching of relevant fluorophores. From this point of view, Struc- tured Illumination Microscopy (SIM), Axial Tomography, Total Internal Reflection Fluorescence Microscopy (TIRFM) and often a combination of these methods are used. To show the high potential of these techniques, measurements on cell-substrate topology as well as on intracellular translocation of the glucose transporter GLUT4 are described. In addition, molecular parameters can be deduced from spectral data, fluorescence lifetimes or non-radiative energy transfer (FRET) between a donor and an acceptor molecule. As an example, FRET between the epidermal growth factor receptor (EGFR) and the growth factor receptor-bound protein 2 (Grb2) is described. Since this interaction, as well as further processes of cellular signaling (e.g., translocation of GLUT4) are sensitive to stimulation by pharmaceutical agents, methods (e.g., TIRFM) are transferred from a fluorescence microscope to a multi-well reader system for simultaneous detection of large cell populations. Keywords: super-resolution microscopy; TIRF; SIM; axial tomography; FRET Citation: Richter, V.; Lanzerstorfer, P.; Weghuber, J.; Schneckenburger, H. Probing Small Distances in Live Cell Imaging. Photonics 2021, 8, 176. https://doi.org/10.3390/ 1. Introduction photonics8060176 Probing small distances is a continuous challenge in life cell imaging. Optical microscopy plays a predominant role in this field as it provides several non-destructive Received: 22 April 2021 techniques for imaging and sensing applications. Methods of transmission, scattering or Accepted: 20 May 2021 fluorescence microscopy have been reported for many years, however, their resolution Published: 21 May 2021 is generally limited to about 200 nm. Only in the last 30 years, this so-called Abbe limit has been overcome by super-resolution techniques, where resolutions below 100 nm Publisher’s Note: MDPI stays neutral have been attained. This offers new insights into many compartments or organelles of a with regard to jurisdictional claims in cell, e.g., nuclei, mitochondria, lysosomes and further vesicles, as outlined in Table1. published maps and institutional afﬁl- Specific techniques, e.g., Total Internal Reflection Fluorescence Microscopy (TIRFM) iations. even allow for selective imaging of cell membranes whose thickness is only about 5 nm [1,2]. Imaging options in the range of only a few nanometers are limited, however, information about the micro-environment of specific molecules, e.g., proteins, can be obtained from sensing additional parameters, e.g., fluorescence spectra or lifetimes. In Copyright: © 2021 by the authors. this context measurement of Förster Resonance Energy Transfer (FRET) [3] from a so- Licensee MDPI, Basel, Switzerland. called donor to an acceptor molecule plays a predominant role not only for fluorescence This article is an open access article staining, but also for measuring intra-molecular or intermolecular distances in the distributed under the terms and nanometer range (for an overview see [4–6]). This manuscript gives an overview on conditions of the Creative Commons high resolution microscopy as well as on nanometer sensing. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Photonics 2021, 8, 176. https://doi.org/10.3390/photonics8060176 https://www.mdpi.com/journal/photonics Photonics 2021, 8, 176 2 of 10

Table 1. Typical size of cell organelles and related super-resolution techniques.

Organelle Diameter (typ.) Technique nucleus 6–12 µm SIM 1 mitochondrium 0.5–1.5 µm lysosome 0.1–1.2 µm microtubule ~25 nm STED/MINFLUX/SMLM 1 Actin ﬁlament ~7 nm membrane 4–5 nm TIRFM protein 3–6 nm 1 Techniques are further described in Section2.

2. Microscopy The lateral resolution ∆x of a common (wide-ﬁeld) microscope is deﬁned either by the Abbe criterion (for coherent illumination)

∆x ≥ λ/2AN (1)

or by the Rayleigh criterion (for a luminescent object or incoherent illumination)

∆x = 0.61 λ/AN (2)

with the wavelength λ and the numerical aperture AN of the microscope objective lens. In both cases values around 200 nm are achieved for numerical apertures AN ≥ 1.30 at λ = 500 nm. Often the axial resolution is related to the depth of focus

2 ∆z = n λ/AN (3)

resulting in ∆z ≈ 400 nm for the refractive index n = 1.50, λ = 500 nm and AN = 1.40. In comparison with wide-field microscopy, (confocal or multiphoton) laser scanning microscopy offers numerous advantages concerning contrast and optical sectioning. However, for an increase of resolution in confocal microscopy a pinhole is required that selects only a small part of the diffraction pattern (“Airy disk”) of individual points, thus reducing the detection sensitivity considerably. Alternatively, an array of several pinholes with individual detectors (Airy Scan Microscopy), an ultrasensitive camera chip (image scan microscopy) or a pinhole with a scanning device (Re-Scan Confocal Microscopy [7]) may replace the original pinhole (for an overview see e.g., [8]). These alternatives, however, make the whole setup rather complex and expensive. Therefore, to increase resolution and limit the complexity of the optical system, several super-resolution or super-localization methods have been developed. Structured Illumination Microscopy (SIM) [9–11] with a periodically modulated illumination pattern leads to a resolution enhancement around a factor of 2 in the lateral direction compared to the value given by the Abbe criterion. Here, the sample is illuminated by 2 interfering laser beams creating a sinusoidal pattern (Figure1) that may be rotated to obtain isotropic resolution enhancement in the lateral plane. Images are recorded for at least 3 rotation angles and 3 phases (0, 2π/3, 4π/3) of the interference pattern, and a super-resolution image is calculated from a minimum of 9 individual images. While 2 interfering beams are sufficient for enhanced resolution in the lateral dimension, 3 interfering beams are needed for increasing resolution in all 3 dimensions. It should be mentioned that resolution can be enhanced even by more than a factor 2, if the emission rate of the sample responds non-linearly to the illumination intensity, using e.g., saturation effects or photo-switching of a fluorescent protein [12]. In some cases, especially for larger specimens, Lattice Light Sheet Microscopy, a combination of Light Sheet Microscopy and SIM, is a useful tool for observing subcellular processes in three dimensions. This technique achieves high resolution (∆x = 150 nm, Photonics 2021, 8, 176 3 of 10

Photonics 2021, 8, 176 ∆z = 280 nm) and contrast at high image acquisition speed, thus minimizing damage3 of of 10

cells due to phototoxicity [13,14].

FigureFigure 1. 1.SIM SIM (schematic) (schematic) with with spatial spatial light light modulator modulator SLM SLM (illuminated (illuminated by by an an argon argon ion ion laser), laser), telescope telescope lenses lenses L 1L,L1, 2L,2, tubetube lens lens TL TL and and objective objective lens lens OL. OL. Further Further components components (λ/4 (λ plate,/4 plate, pinhole pinhole plate, plate, polarizer, polarizer, dichroic dichroic mirror mirror and cameraand camera are omitted).are omitted). If the If two the interferingtwo interfering laser laser beams beams are incidentare incident close close to theto the edges edges of aof high a high aperture aperture OL, OL, SIM SIM may may be be combined combined with Total Internal Reflection Microscopy (TIRFM), as reported below. Reproduced from [15] with modifications. with Total Internal Reﬂection Microscopy (TIRFM), as reported below. Reproduced from [15] with modiﬁcations.

TotalTotal Internal Internal ReflectionReflection Fluorescence Microscopy Microscopy (TIRFM) (TIRFM) isis a wide-field a wide-field technique technique that thatpermits permits selective selective detection detection of ofsurfaces, surfaces, e. e.g.,g., plasma plasma membranes. When aa lightlight beam beam 1 propagatingpropagating through through a mediuma medium of of refractive refractive index index n1 (e.g.,n (e.g., glass) glass) meets meets a second a second medium medium of refractiveof refractive index index n2 < nn12(e.g., < n1 cell(e.g., membrane cell membrane or cytoplasm), or cytoplasm), total internal total reflection internal occursreflection at alloccurs angles at of all incidence angles Θof, whichincidence are greaterΘ, which than are a critical greater angle thanΘ ac =critical arcsin (nangle2/n 1Θ).c Thereby, = arcsin an(n evanescent2/n1). Thereby, electromagnetic an evanescent field electromagnetic is generated field that penetratesis generated about that 100penetrates nm into about the sample100 nm and into permits the selectivesample excitationand permits of membrane selective proximalexcitation fluorophores. of membrane “Prism-type proximal TIRF”,fluorophores. based on “Prism-type light incidence TIRF via”, a based glass oron quartz light incidence prism whose via refractivea glass or index quartz exceeds prism thatwhose of the refractive specimen, index has beenexceeds used that for liveof the cell measurementsspecimen, has forbeen almost used 40 for years live [1 ,2cell]. Bymeasurements variation of thefor anglealmost of 40 incidence years [1,2]. (“VA-TIRFM”), By variation an of axial the resolutionangle of incidence of only a(“VA- few nanometersTIRFM”), an becomes axial resolution possible, whichof only permits a few nanometers studies of cell-substrate becomes possible, topology, which e.g., permits upon applicationstudies of cell-substrate of cytotoxic ortopology, phototoxic e.g., agents upon a orpplication in studies of ofcytotoxic diseases. or Anphototoxic illumination agents deviceor in studies (microscope of diseases. condenser) An illumination for variable-angle device TIRFM(microscope is depicted condenser) in Figure for variable-2a, an applicationangle TIRFM to Chineseis depicted Hamster in Figure Ovary 2a, an (CHO) application cells expressing to Chinese a membrane-associatedHamster Ovary (CHO) Greencells expressing Fluorescence a membrane-associated Protein (GFP) in Figure Green2b,c. WhileFluorescence the penetration Protein (GFP) depth in d Figure ( Θ) of the2b,c. evanescentWhile the electromagneticpenetration depth wave d (Θ depends) of the onevanescent the angle electromagnetic of incidence Θ, wave the fluorescence depends on intensitythe angle of theof TIRFMincidence signal Θ, forthe a homogenouslyfluorescence intensity fluorescent of layerthe ofTIRFM thickness signal t can for be a calculatedhomogenously as fluorescent layer of thickness t can be calculated as −∆/d(Θ) IF (Θ) = A T(Θ) t e (4) IF (Θ) = A T(Θ) t e−Δ/d(Θ) (4) with a constant A, a transmission factor T(Θ) through the cell-substrate interface, a penetra- tionwith depth a constant d(Θ) of theA, evanescenta transmission wave factor and a distanceT(Θ) through∆ between the acell-substrate cell and its substrate interface, [16]. a Variationpenetration of the depth angle d(Θ)permits of the evanescent imaging of wave this distance and a distance∆ for the Δ wholebetween specimen a cell and (cell- its substratesubstrate topology), [16]. Variation and arbitrary of the angle objective Θ permits lenses imaging can be used of this for distance image detection. Δ for the whole specimenIn “Objective-type (cell-substrate TIRF”, topology), which and became arbitrary available objective 20 yearslenses latercan be than used prism-type for image TIRFM,detection. the illumination beam is focused close to the edge of the aperture of a microscope objective lens with very high numerical aperture, so that illumination occurs under total internal reflection [17]. Alternatively, the focused spot may be scanned in a circular orbit for homogeneous illumination of the sample under various directions during short exposure times (HILO technique [18]). Objective-type TIRF can be combined with structured illumination microscopy (SIM) using up to 6 interfering laser beams to achieve super-resolution in three dimensions (see Figure1)[ 18–21]. A comparison of SIM and TIRF-SIM is shown in Figure3 for a Chinese Hamster Ovary (CHO) cell transfected with GFP-tagged glucose transporter 4 (GLUT4). Insulin as well as insulin-mimetic compounds, e.g., tannic acid or an extract of Bellis perennis (common daisy) [22], are presently used as stimulating agents to

Figure 2. Condenser for TIR illumination under variable angle (schematic, (a)); TIRFM image of CHO cells expressing a membrane-associated green Fluorescent Protein (GFP) recorded at Θ = 66° (b); cell-substrate distances in the range of 0–

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Figure 1. SIM (schematic) with spatial light modulator SLM (illuminated by an argon ion laser), telescope lenses L1, L2, tube lens TL and objective lens OL. Further components (λ/4 plate, pinhole plate, polarizer, dichroic mirror and camera are omitted). If the two interfering laser beams are incident close to the edges of a high aperture OL, SIM may be combined with Total Internal Reflection Microscopy (TIRFM), as reported below. Reproduced from [15] with modifications.

Total Internal Reflection Fluorescence Microscopy (TIRFM) is a wide-field technique that permits selective detection of surfaces, e.g., plasma membranes. When a light beam propagating through a medium of refractive index n1 (e.g., glass) meets a second medium of refractive index n2 < n1 (e.g., cell membrane or cytoplasm), total internal reflection occurs at all angles of incidence Θ, which are greater than a critical angle Θc = arcsin (n2/n1). Thereby, an evanescent electromagnetic field is generated that penetrates about 100 nm into the sample and permits selective excitation of membrane proximal fluorophores. “Prism-type TIRF”, based on light incidence via a glass or quartz prism whose refractive index exceeds that of the specimen, has been used for live cell Photonics 2021, 8, 176 measurements for almost 40 years [1,2]. By variation of the angle of incidence (“VA-4 of 10 TIRFM”), an axial resolution of only a few nanometers becomes possible, which permits studies of cell-substrate topology, e.g., upon application of cytotoxic or phototoxic agents or in studies of diseases. An illumination device (microscope condenser) for variable- 400 nm calculated from VA-TIRFM according to Equation (1) and shown by color code (c) (excitation wavelength: 476 nm, detection range: λ ≥ 490 nm;angle scale TIRFMbar: 20 µm). is depicted in Figure 2a, an application to Chinese Hamster Ovary (CHO) cells expressing a membrane-associated Green Fluorescence Protein (GFP) in Figure 2b,c. WhileIn the “Objective-type penetration depth TIRF”, d ( Θwhich) of the became evanescent available electromagnetic 20 years later wave than depends prism-type on Photonics 2021, 8, 176 theTIRFM, angle the of illumination incidence beamΘ, the is focusedfluorescence close tointensity the edge ofof thethe aperture TIRFM of signal a microscope 4for of 10a homogenouslyobjective lens with fluorescent very high layer numerical of thickness apertu t canre, beso calculatedthat illumination as occurs under total

internal reflection [17]. Alternatively,IF (Θ the) = foA cusedT(Θ) t spote−Δ/d( mayΘ) be scanned in a circular orbit(4) for homogeneous illumination of the sample under various directions during short triggerwithexposure a translocationconstant times A,(HILO ofa GLUT4transmission technique from intracellular[18]).factor ObjeT(Θ)ctive-type compartmentsthrough theTIRF cell-substrate to can the plasmabe combined membrane,interface, with a aspenetrationstructured further described illuminationdepth d( inΘ) line ofmicroscopy the 209 evanescent ff.. As (SIM) depicted waveusing Figure upand to a3 6 distance(left), interfering we Δ observed between laser beams ﬂuorescencea cell to andachieve its arisingsubstratesuper-resolution primarily [16]. Variation fromin three small of dimensionsthe vesicles angle byΘ (seepermits SIM. Fi Whengure imaging 1) we [18–21]. measured of this A distance comparison the plasma Δ for membraneof the SIM whole and almost exclusively by TIRF-SIM (Figure3, right), we observed a more even distribution of specimenTIRF-SIM (cell-substrate is shown in Figure topology), 3 for aand Chines arbitre Hamsterary objective Ovary lenses (CHO) can ce bell transfectedused for image with ﬂuorescence intensity over the whole cell surface. detection.GFP-tagged glucose transporter 4 (GLUT4). Insulin as well as insulin-mimetic compounds, e.g., tannic acid or an extract of Bellis perennis (common daisy) [22], are presently used as stimulating agents to trigger translocation of GLUT4 from intracellular compartments to the plasma membrane, as further described in line 209 ff.. As depicted Figure 3 (left), we observed fluorescence arising primarily from small vesicles by SIM. When we measured the plasma membrane almost exclusively by TIRF-SIM (Figure 3, right), we observed a more even distribution of fluorescence intensity over the whole cell surface. An alternative method to TIRFM deserves mentioning. It is called “Supercritical angle fluorescence microscopy” (SAF) and is based on full-field excitation of the sample and selective detection of fluorescence above the critical angle Θc [23,24]. A main Figure 2. Condenser for TIR illumination under variable angle (schematic, (a)); TIRFM image of CHO cells expressing Figure 2. Condenser for TIRadvantage illumination of un thisder method variable is angle that (schematic,membrane ( aassociated)); TIRFM image fluorescence of CHO (abovecells expressing Θc) and awhole ◦ amembrane-associated membrane-associated green green cellFluorescent Fluorescent fluorescence Protein Protein (below (GFP) (GFP) recordedΘ recordedc) can atbe at Θ observedΘ = =66° 66 (b();b );simultaneously,cell-substrate cell-substrate distances distances but a in disadvantage inthe the range range of of0– is that 0–400 nm calculated from VA-TIRFMthe total according light exposure to Equation is higher (1) and than shown for by TIRFM color code experiments. (c) (excitation Also wavelength: variation 476 of nm,the angle detection range: λ ≥ 490 nm;Θ scale is difficult bar: 20 µ m).to realize.

Figure 3. Comparison of SIM (left) and SIM-TIRFM (right): CHO-K1-hIR-myc-GLUT4-GFP cell, Figure 3. Comparison of SIM (left) and SIM-TIRFM (right): CHO-K1-hIR-myc-GLUT4-GFP cell, excitation wavelength: λex = 488 nm; detection range: λD ≥ 515 nm; Plan Neofluar 40×/1.30 oil excitation wavelength: λ = 488 nm; detection range: λ ≥ 515 nm; Plan Neofluar 40×/1.30 oil immersion lens (left) or exPlan Apochromat 63×/1.46 oil immersionD objective lens (right). GFP × immersionfluorescence lens arising (left) orfrom Plan intracellular Apochromat vesicles 63 /1.46 (left) oil or immersionthe whole plasma objective membrane lens (right (right). GFP). fluo- rescenceReproduced arising from from [15] intracellular with modifications. vesicles (left ) or the whole plasma membrane (right). Reproduced from [15] with modifications. Stimulated Emission Depletion (STED) microscopy is a laser scanning technique that An alternative method to TIRFM deserves mentioning. It is called “Supercritical angle overcomes the diffraction limited resolution of confocal and multiphoton microscopes. fluorescence microscopy” (SAF) and is based on full-field excitation of the sample and The enhancement of resolution is due to suppression of fluorescence of the dye molecules selective detection of fluorescence above the critical angle Θc [23,24]. A main advantage of in the outer regions of a diffraction limited illumination spot by stimulated emission using this method is that membrane associated fluorescence (above Θc) and whole cell fluores- a (second) donut shaped laser beam. While a resolution of 30–70 nm can thus be achieved cence (below Θc) can be observed simultaneously, but a disadvantage is that the total light [25], the irradiance exceeds that of a conventional fluorescence microscope by a factor 104– exposure is higher than for TIRFM experiments. Also variation of the angle Θ is difficult 105 and may cause severe damages to living specimens. This problem was minimized with to realize. the introduction of MINFLUX nanoscopy, a technique based on localization and tracking Stimulated Emission Depletion (STED) microscopy is a laser scanning technique that overcomes the diffraction limited resolution of confocal and multiphoton microscopes. The enhancement of resolution is due to suppression of fluorescence of the dye molecules in the outer regions of a diffraction limited illumination spot by stimulated emission using a (second) donut shaped laser beam. While a resolution of 30–70 nm can thus be achieved [25], the irradiance exceeds that of a conventional fluorescence microscope by a factor 104–105 and may cause severe damages to living specimens. This problem was minimized with the introduction of MINFLUX nanoscopy, a technique based on localization and tracking Photonics 2021, 8, 176 5 of 10

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of single molecules in the intensity minimum of a donut-shaped laser beam. MINFLUX achieves nanometerof single resolution molecules (isotropic: in the intensity ≥ 2 nm) minimum at moderate of alight donut-shaped exposure, laserwhich beam. is MINFLUX comparable to achievesconfocal nanometerlaser scanning resolution microscopy (isotropic: [26]. ≥ 2 nm) at moderate light exposure, which is Super-resolutioncomparable may to further confocal result laser from scanning single microscopy molecule localization [26]. microscopy (SMLM) within a Super-resolutionthin illuminated maylayer furtherof a sample result from[27–30]. single If a moleculesingle molecule localization is microscopy detected n times, its localization can be determined with a precision Δx = Δx0/√n with Δx0 (SMLM) within a thin illuminated layer of a sample [27–30]. If a single√ molecule is detected ~200 nm accordingn times, to the its localizationRayleigh criterion. can be determined Therefore, a with precision a precision of localization∆x = ∆x0/ Δxn = with 20 ∆x0 ~200 nm nm results fromaccording n = 100 and to Δ thex = Rayleigh10 nm from criterion. n = 400. Methods Therefore, based a precision on super-localization of localization ∆x = 20 nm microscopy includeresults Stochastic from n = Optical 100 and Reconstruction∆x = 10 nm fromMicroscopy n = 400. (STORM) Methods, Photoactivation based on super-localization Localization Microscopymicroscopy (PALM) include andStochastic related techniques. Optical Reconstruction Generally, SMLM Microscopy techniques (STORM) need, Photoactivation an irradiance, Localizationwhich is about Microscopy 100 times (PALM) largerand than related for conventional techniques. Generally, microscopy SMLM and techniquesa need prolonged exposurean irradiance, time of at which least a is few about second 100s, times so that larger the risk than of for cell conventional damage is again microscopy and a very high. prolonged exposure time of at least a few seconds, so that the risk of cell damage is again Most microscopyvery high. techniques can also be combined with Axial Tomography to overcome the anisotropyMost microscopy of optical resolution techniques. As can shown also be by combined the point with spreadAxial function Tomography in to overcome Figure 4a the thelateral anisotropy resolution of opticalis generall resolution.y higher As than shown the byaxial the resolution. point spread However, function in Figure4a after rotation alwaysthe lateral the resolutionoptimal (lateral) is generally resolu highertion can than be used the axial as depicted resolution. in Figure However, 4b. after rotation A combinationalways with thesuper-resolution optimal (lateral) micros resolutioncopy cangives be usedan extremely as depicted high in Figure isotropic4b. A combination resolution, e.g.,with about super-resolution 100 nm in the microscopy case of SIM gives (Figure anextremely 4c). Rotation high of isotropic samples resolution, needs e.g., about specific sample100 holders nm in and the preparation case of SIM techni (Figureques,4c). Rotatione.g., cells ofembedded samples needsin a gel specific within samplea holders rotatable capillary,and preparation whose refractive techniques, index e.g., can cells be adapted embedded to inthat a gel of withinwater aor rotatable oil when capillary, whose using an immersionrefractive lens indexof high can magnification be adapted to[31]. that A of sample water holder or oil when with fine using tuning an immersion of lens of ◦ rotation angleshigh over magnification 360° has been [31 reported]. A sample previously holder with[32]. fineIt can tuning be easily of rotation adapted angles to a over 360 has great variety ofbeen common reported light previously microscopes [32 ].and It can is suitable be easily for adapted various to applications a great variety in life of common light science. microscopes and is suitable for various applications in life science.

Figure 4. PointFigure spread 4. Point function spread for function high resolution for high wide-ﬁeld resolution microscopywide-field microscopy (a), axial tomography (a), axial tomography (b) and a combination of (b) and a combination of axial tomography with Structured Illumination Microscopy (SIM) (c). axial tomography with Structured Illumination Microscopy (SIM) (c).

In every opticalIn system, every opticallight emitted system, from light each emitted point in from the eachobject point is convolved in the object with is convolved the point spreadwith function the point (PSF) spread of the system function to (PSF)produce of the the final system image. to produceThis convolution the ﬁnal image. This causes points convolutionin the object causes plane points to become in the objectblurred plane regions to become in the blurred image regionsplane, thus in the image plane, degrading imagingthus degrading quality. imagingDeconvolution quality. is Deconvolutiona post-acquisitionis a post-acquisition image processing image processing technique usedtechnique to reverse used this to process reverse to this partially process restore to partially lost resolution restore lost and resolution improve and improve contrast. However,contrast. as with However, all analytical as with tools, all analytical it must be tools, applied it must with be care applied to maximize with care to maximize reliability and reliabilityavoid artifacts and avoid[33,34]. artifacts [33,34].

3. Sensing 3. Sensing Depending onDepending the method on of themicroscopy, method of re microscopy,solution is limited resolution to 10–100 is limited nm, toi.e., 10–100 nm, i.e., smaller structuressmaller cannot structures be resolved cannot in be an resolved image. in However, an image. it However, is well known it is well that known that flu- fluorescence spectraorescence as well spectra as fluorescence as well as fluorescencelifetimes depend lifetimes on the depend microenvironment on the microenvironment of of a relevant fluorophore, including pH, viscosity, and polarity [35,36]. In particular, after

PhotonicsPhotonics2021 2021,8 8, 176 6 of 10 , , 176 6 of 10

a relevant fluorophore, including pH, viscosity, and polarity [35,36]. In particular, after excitation of a molecular species by a short light pulse its ﬂuorescence often shows an excitation of a molecular species by a short light pulse its fluorescence often shows an exponential decay with the time constant exponential decay with the time constant τ −1 =τ (k =F (k+Fk +nr k)nr)−1 (5)(5)

wherewhere kkFF representsrepresents thethe raterate ofof radiativeradiative (ﬂuorescent)(fluorescent) transitionstransitions andand kknrnr thatthat ofof non-non- radiativeradiative transitionstransitions fromfrom anan excitedexcited molecularmolecular state.state. kknrnr depends onon variousvarious parameters,parameters, e.g.,e.g., molecularmolecular conformation, conformation, intersystem intersystem crossing crossing between between excited excited singlet singlet and triplet and triplet states orstates the molecule’sor the molecule’s microenvironment. microenvironment Coming. Coming back to back the stimulus-dependent to the stimulus-dependent translo- cationtranslocation of GLUT4 of GLUT4 from intracellular from intracellular storage st compartmentsorage compartments to the to plasma the plasma membrane membrane (see Figure(see Figure3 ), we 3), measured we measured the ﬂuorescence the fluorescen lifetimece oflifetime GLUT4-GFP of GLUT4-GFP prior to and prior subsequent to and tosubsequent stimulation to withstimulation insulin orwith the insulin insulin-mimetic or the insulin-mimetic substances tannic substances acid or Bellistannic perennis acid or extract.Bellis perennis Here, weextract. illuminated Here, we either illuminated whole cells either or theirwhole plasma cells or membranes their plasma at membranes an angle of 62at◦ an(epi-illumination) angle of 62° (epi-illumination) or 66◦ (TIRF), asor depicted66° (TIRF), in as Figure depicted5. in Figure 5.

FigureFigure 5.5.Fluorescence Fluorescence lifetimelifetime ofof CHO-K1CHO-K1 hIR/GLUT4-myc-GFPhIR/GLUT4-myc-GFP cells priorprior toto (left(left columns)columns) andand subsequentsubsequent toto (right(right columns)columns) 3030 min min stimulation stimulation with with insulin, insulin, tannic tannic acid acid or orBellis Bellis perennis perennisextract. extract. Results Results show show means means± ± standardstandard deviationsdeviations ofof 1313 (insulin)(insulin) oror 1414 (tannic(tannic acid,acid,Bellis Bellis perennisperennis)) cell cell specimens specimens upon upon whole whole cell cell (“EPI”) (“EPI”) or or TIRF TIRF illumination. illumination. LevelsLevels ofof significance:significance: ** pp << 0.01,0.01, ******** pp << 0.0001,0.0001, andand n.s.n.s. nono significantsignificant differencedifference forfor comparisoncomparison ofof epi-epi- andand TIRTIR illuminationillumination (reproduced from [15], Supplementary Information, with modifications). (reproduced from [15], Supplementary Information, with modifications).

AccordingAccording toto Figure Figure5, fluorescence5, fluorescence lifetimes lifetimes were were around around 2.2 ns 2.2 upon ns TIRF upon illumi- TIRF nationillumination and remained and remained almost constantalmost constant after stimulation. after stimulation. These values These reflect values membrane reflect associatedmembrane GLUT4-GFPassociated GLUT4-GFP fluorescence fluorescence with increasing with intensity, increasing but intensity, almost constant but almost life- timeconstant after lifetime stimulation. after stimulation. In contrast, fluorescenceIn contrast, fluorescence lifetimes of 2.5–2.8lifetimes ns of within 2.5–2.8 whole ns within cells decreasedwhole cells upon decreased stimulation upon to stimulation 2.3–2.4 ns, thus to 2.3–2.4 approaching ns, thus the valuesapproaching measured the withinvalues themeasured plasma within membrane. the plasma This decreasemembrane. was This significant decrease for was stimulation significant by for insulin stimulation and Bel- by lisinsulin perennis andextract Bellis andperennis indicated extract a trend and forindicated tannic acid.a trend Therefore, for tannic fluorescence acid. Therefore, lifetime mayfluorescence represent lifetime a quantitative may represent molecular a quanti parametertative molecular for fluorescence parameter re-distribution for fluorescence from intracellularre-distribution vesicles from tointracellular the plasma vesicles membrane to the as observedplasma membrane in the SIM/TIRF-SIM as observed images. in the InSIM/TIRF-SIM this context it images. should beIn this mentioned context thatit should some be shortening mentioned of GFPthat some lifetime shortening in close vicinity of GFP tolifetime a cell-glass in close interface vicinity has to beena cell-glass reported interface in the literaturehas been reported [37]. in the literature [37]. InIn thethe casecase ofof non-radiativenon-radiative energyenergy transfertransfer fromfrom anan excitedexcited donordonor moleculemolecule toto anan acceptoracceptor moleculemolecule (FRET(FRET [[3])3]) thethe energyenergy transfertransfer raterate k kETET contributescontributes toto thetherate rate k knrnr andand maymay causecause aa pronouncedpronounced decreasedecrease ofof thethe fluorescencefluorescence lifetimelifetime ττ ofof thethe donordonor molecule.molecule. ThisThis effecteffect has has been been used used frequently frequently to measureto measure changes changes of molecular of molecular conformations conformations (“intramolecular(“intramolecular FRET”) FRET”) as well as aswell intermolecular as intermolecular interactions interactions of the optical of the transitionoptical transition dipoles ofdipoles specific of specific chromophores. chromophores. Examples Examples of intramolecular of intramolecular FRET FRET are calcium are calcium signaling signaling [38] or[38] cyclic or cyclic AMP AMP signaling signaling using using a so-called a so-called EPAC EPAC sensor sensor ([39] ([39] and referencesand references therein). therein). Ex- amplesExamples for for intermolecular intermolecular FRET FRET are are manifold. manifold. They They include include intrinsic intrinsic fluorophoresfluorophores asas wellwell asas fluorescentfluorescent membranemembrane probesprobes (for(for aa reviewreview seesee [[6]),6]), butbut sincesince thethe introductionintroduction ofof GFPGFP [40[40]] and and its its variants variants for for the the blue, blue, yellow yellow and and red spectralred spectral range, range, cellular cellular proteins proteins fused withfused one with of theseone of GFP these variants GFP variants are often are used often as energyused as donors energy or donors acceptors. or acceptors. Relevant topicsRelevant include topics interactions include interactions of cellular growth of cellul factorar growth proteins factor [41,42 proteins] as well caspase[41,42] as driven well

Photonics 2021, 8, 176 7 of 10

caspase driven molecular interactions upon apoptosis [43,44]. Furthermore, FRET experiments are used increasingly for studies of pathogenesis of various diseases, e.g., tumors [45] or Alzheimer’s disease [46]. Altogether, FRET can be evaluated on the basis of spectral data (e.g., ratio measurements of donor and acceptor fluorescence), fluorescence lifetimes and polarization measurements, which are of particular interest, if donors and acceptors represent the same type of molecules (homo-FRET [47]). A major restriction of ratio measurements may be the spectral overlap of donor and acceptor emission (which requires some deconvolution and background subtraction) and the often Photonics 2021, 8, 176 unknown fluorescence quantum yields, while the main problem of fluorescence lifetime7 of 10 measurements may be the appropriate reference of a sample without acceptor or with a photo-bleached acceptor (for further details see [4–6]. molecularWhen interactions FRET is combined upon apoptosis with TIR-illumi [43,44].nation, Furthermore, cell membranes FRET experiments and protein-protein are used increasinglyinteractions for close studies to ofthe pathogenesis plasma membrane of various can diseases, be examined e.g., tumors selectively. [45] or Alzheimer’s A recent diseaseapplication [46]. of Altogether, TIR-FRET FRET is depicted can be evaluatedin Figure on6 showing the basis the of spectralinteraction data between (e.g., ratio the measurementsepidermal growth of donor factor and recept acceptoror (EGFR), fluorescence), fused with fluorescence Cyan Fluorescent lifetimes Protein and polarization (CFP), and measurements,the growth factor which receptor-bound are of particular protein interest, 2 (Grb2), if donors fused and with acceptors Yellow representFluorescent the Protein same type(YFP). of moleculesThe EGFR (homo-FRET regulates important [47]). A major pathways restriction such ofas ratiogrowth, measurements survival, proliferation may be the spectraland differentiation overlap of donor in mammalian and acceptor cells, emission and has (which become requires a major some drug deconvolution target, as EGFR and backgroundsignaling issubtraction) critical for andthe thedevelopment often unknown of many fluorescence types of quantum cancer [48]. yields, In whileFigure the 6, mainmembrane problem specific of fluorescence donor and lifetime acceptor measurements fluorescence may is depicted be the appropriate (top), and reference fluorescence of a samplelifetime without images acceptor of the donor or with EGFR-CFP a photo-bleached are shown acceptor in absence (for further and in details presence see [ 4of–6 ].the acceptorWhen Grb2-YFP FRET is combined (bottom). with Pronounced TIR-illumination, shortening cell membranesof the fluorescence and protein-protein lifetime is interactionsobserved in close the tosecond the plasma case due membrane to FRET. can Furthermore, be examined the selectively. efficiency A recentof energy application transfer offrom TIR-FRET EGFR-CFP is depicted to Grb2-YFP in Figure increased6 showing after the stimulation interaction betweenof the cells the with epidermal the epidermal growth factorgrowth receptor factor (EGFR),(EGF) [49]. fused with Cyan Fluorescent Protein (CFP), and the growth factor receptor-boundAs a valuable protein contribution 2 (Grb2), fusedto the with growing Yellow field Fluorescent of high throughput Protein (YFP). screening, The EGFR the regulatesTIR-FRET important technique pathways was transferred such as growth, from a survival,fluorescence proliferation microscope and to differentiation a multi-well inreader mammalian system cells,for simultaneous and has become detection a major of druglarge target,cell populations as EGFR signaling grown in is adapted critical formicrotiter the development plates. A 96-well of many plate types reported of cancer earl [48ier]. [50] In Figure was modified6, membrane for this specific purpose, donor and andin addition acceptor to fluorescence TIR-FRET experiments is depicted(top), this fluorescence and fluorescence reader lifetime was used images to quantify of the donor GLUT EGFR-CFP4 translocation are shown from inintracellular absence and storage in presence compartments of the acceptor to the Grb2-YFPplasma membrane. (bottom). PronouncedStimulation shorteningby insulin ofcould the fluorescencethus be measured lifetime quantitatively is observed in in the a secondlarge concentration case due to FRET.range Furthermore, between 10–12 the and efficiency 10–6 ofmol/L energy [51], transfer and insulin from EGFR-CFP mimetic compounds to Grb2-YFP are increased further afterevaluated stimulation in view of of the their cells efficacy with the for epidermal treatment growth of type factor 2 diabetes (EGF) mellitus. [49].

Figure 6. TIR ﬂuorescence images of HeLa cells transfected with EGFR-CFP and Grb2-YFP in the spectral ranges of donor (450–490 nm) and acceptor (λ ≥ 510 nm) emission (top); ﬂuorescence

lifetimes of the donor EGFR-CFP in absence (bottom, left) and presence (bottom, right) of the acceptor; excitation wavelength: 420–440 nm; image size: 60 µm × 60 µm (top), 100 µm × 100 µm (bottom). Reproduced from [39] with modiﬁcations.

As a valuable contribution to the growing field of high throughput screening, the TIR-FRET technique was transferred from a fluorescence microscope to a multi-well reader system for simultaneous detection of large cell populations grown in adapted microtiter plates. A 96-well plate reported earlier [50] was modified for this purpose, and in addition Photonics 2021, 8, 176 8 of 10

to TIR-FRET experiments this ﬂuorescence reader was used to quantify GLUT 4 translocation from intracellular storage compartments to the plasma membrane. Stimulation by insulin could thus be measured quantitatively in a large concentration range between 10–12 and 10–6 mol/L [51], and insulin mimetic compounds are further evaluated in view of their efﬁcacy for treatment of type 2 diabetes mellitus.

4. Discussion Methods for probing small distances in life cell imaging—including super-resolution microscopy and molecular sensing—are reported. Super-resolution techniques, however, should be applied with care in order to avoid photochemical damages of the cells as well as photobleaching of fluorescent specimens. Presently, SIM with a lateral resolution around 100 nm seems to fulfill the requirement of low or moderate light exposure. MINFLUX technology with a resolution of only a few nanometers may be a promising alternative in the near future. For cell membrane studies with high axial resolution, TIRFM appears to be the method of choice, since by variation of the angle of incidence (VA-TIRFM) cell- substrate distances can be determined with a precision of a few nanometers. Prism-type TIRFM may be advantageous for selection of a wide angular range in comparison to objective-type TIRFM. In addition, prism-type TIRFM has the advantage that any kind of objective lens (with arbitrary aperture and magnification) can be used. Prism-type TIRF technology can also be applied in new fields, e.g., fluorescence screening of larger cell collectives in microtiter plates, which has a high potential in pharmaceutical sciences. In addition, various combinations of methods, e.g., TIRF-SIM with a high lateral and axial resolution, as well a combination of axial tomography with further super-resolution methods, appear promising. Whenever small dimensions within a sample cannot be visualized in a microscope, fluorescent molecules can be localized and molecular distances can be estimated on the basis of spectral data, fluorescence lifetimes or FRET between a donor and an acceptor molecule. FRET data (spectra, fluorescence lifetimes or FRET efficiencies) can also be displayed as high resolution images.

Author Contributions: Conceptualization, H.S.; methodology, H.S., V.R. and P.L.; software, V.R. and P.L.; validation, H.S., V.R. and P.L.; investigation, V.R.; resources, H.S.; data curation, V.R.; writing— original draft preparation, H.S. and V.R.; writing—review and editing, H.S. and J.W.; visualization, V.R.; supervision, H.S. and J.W.; funding acquisition, H.S. and J.W. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Christian Doppler Forschungsgesellschaft (Josef Ressel Center for Phytogenic Drug Research, Wels, Austria) as well as by the province of Upper Austria as part of the FH Upper Austria Center of Excellence for Technological Innovation in Medicine (TIMed CENTER). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The authors thank Michael Wagner for his cooperation and Claudia Hintze for skillful technical assistance. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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