Confocal Microscopy

Abstract

Confocal microscopy is nowadays one of the most extensively used tools for digital imaging. The advances in instrumental setup and fluorescence labelling strategies improve and broaden the possible experimental applications. Moreover, the implementation of solutions able to break the diffraction barrier coupled to confocal instruments brings up the study of the samples at super‐resolution level. This continuous evolution ensures confocal microscopy as a key platform in life science research.

Key Concepts

  • Components of confocal laser scanning and spinning disk microscopes.
  • Fluorescence and fluorescent labelling tools.
  • Confocal microscopy setup and main applications review.
  • Confocal microscopy applications: reflection, subcellular localisation, dynamic studies and molecular interactions.
  • Super‐resolution approaches in confocal microscopy.

Keywords: confocal microscopy; laser scanning; spinning disk; pinhole; resolution; detectors; fluorescence; fluorochromes; fluorescent proteins; localisation; molecular interaction; dynamic studies; super‐resolution; software

Figure 1. Excitation and an example of the effect of different pinhole sizes on the sample. (A) Excitation of the fluorophore on a widefield or confocal microscope. (B) How emission on a confocal microscope would appear as detected through the pinhole of various sizes: (a) 4 Airy units (AU); (b) 2 AU; and (c) an optimal pinhole, around 1 AU.
Figure 2. The excitation and emission path to understand how CLSM works. (a) In the excitation path, the acousto‐optical tunable filters (AOTF) selects the laser line and its intensity to direct the beam through dichroic mirror to scanner. The X‐galvanometer mirror rapidly moves horizontally along the x‐axis to scan every pixel, the Y‐galvanometer mirror moves to change vertically the y‐axis, and again the X‐galvanometer mirror rapidly scans horizontally a new line of the specimen, to get complete scanning of the field of interest; (b) in the emission path, the fluorescence emission of every pixel is imaged onto a detector through the galvanometer mirrors and the dichroic. Then the pinhole blocks the fluorescence from out‐of‐focus light, only allowing light from the focused plane to be directed onto the detector; (c) the excitation and emission path in a CLSM.
Figure 3. Applications for studies about molecular interactions. (A) (a) FRET Representation of the principle of FRET: if two molecules labelled with overlapping and properly oriented fluorochromes (D and A, donor and acceptor) are very close (<10 nm), the excitation of the donor molecule (D′) can lead to the excitation of the acceptor one (A′) and the emission of the fluorescence from A′. (b) Representations of fluorescence variations in FRET‐complex molecules (D′ and A′) before (pre‐bleach) and after (post‐bleach) the application of high power laser at the specific λ for A′ excitation (bright green beam) that causes the acceptor photobleaching (red‐white star). The excitation of FRET complex with the appropriate λ (blue and green beams for D′ and A′, respectively) results in D′ only fluorescence emission since there is no energy transfer to the A′ molecule. (c) Representations of the sensitised emission FRET method. In conditions of no FRET, D excitation does not alter A excitation or emission. If both molecules are close enough and set up a FRET complex, the return to the ground state after D′ excitation (blue beam) results in A′ excitation. The consequences of the energy transfer from D′ to A′, are the decrease in D′ emission and the increase of A′ emission. (B) FLIM: Representations of FLIM measurements. (a) Donor fluorescence lifetime variations (single channel studies) according to different FRET situations: no FRET, with higher lifetime and low or high FRET, with shorter donor lifetimes (faster decay). (b) Effect of FRET on the fluorescence lifetime of a molecular complex or double‐tagged FRET experiment, showing the inverse result to donor case: the occurrence of high FRET implies a higher lifetime, whereas no FRET involves a faster decay. (C) BiFC: Representation of the principle of BiFC technique: reconstruction of the fluorescent properties of a fragmented fluorescent protein (e.g. YFP), each part fused to the protein of interest, in conditions of bimolecular complementation: molecular proximity (<10 nm), appropriate fragment orientation and expression time and controlled environmental conditions.
Figure 4. Applications for dynamic studies. (A) (a) FRAP: Representation of a FRAP experiment: After a period of image acquisition of cells expressing the labelled molecule of interest (pre‐bleach), an intense laser beam bleaches irreversibly the fluorochromes in a selected ROI. The recording of the fluorescence recovery in the ROI over time (post‐bleach period) allows the evaluation of dynamic parameters of the molecules. (b) Representative plot of the signal evolution in a FRAP experiment. The recovery of the ROI fluorescence intensity after the bleaching cycles (I0) reaches a maximum plateau value (IR) that allows the differentiation of two sets of molecules according to their mobility, the mobile (Mf, from I0 to IR) and immobile fractions (IMf, from IR to Ii). The intensity values for correction and normalisation come from the acquisition decay curve (from a ROI for experimental photobleaching correction), a ROI for the evaluation of total fluorescence in the sample and a ROI for background determination in the proximity of the cell. (B) (a) FLIP: Representation of a FLIP experiment: Repetitive bleaching in a selected ROI and simultaneous measurement of fluorescence intensity variations in regions outside the bleaching area. The application of repetitive bleaching cycles causes a general decrease in the cellular fluorescence due to the bleaching of new populations of labelled molecules that move into the bleaching ROI. (b) Representative plot of the fluorescence intensity evolution, that decays exponentially and allow the differentiation of mobile and immobile fractions and even different mobility capacities of the molecules. (C) Optical highlighters. Representations of experiments with proteins with characteristic fluorescent properties. PA (photoactivation) implies the activation (usually with high power laser line at 405 nm) of the fluorochromes of a selected ROI and the imaging of the fluorescence intensity variations after excitation with the proper fluorophore λ (e.g. 488 nm). The dynamic of the molecules changes into the activated ROI from high fluorescence intensity to a decrease caused by their movement by diffusion or transport to other cellular regions. To compensate the plausible signal dilution, it is possible to repeat several times the ROI photoactivation step. In the case of PS (photoswitch) assays, the use of proteins able to alternate between nonfluorescent and fluorescent (dark) states without photobleaching allow the use of repeated ‘on‐off’ cycles to study the dynamic properties within the same ROI with very low‐intensity loss. PC (photoconversion) experiments exploit the ability of PC proteins to change the emission λ depending on the selected illumination. This process favours the discrimination between subsets of the same population of molecules (or even organelles such as mitochondria) and the recording of their dynamic behaviour. All of these optical highlighters are an efficient tool in single‐molecule localisation applications. (D) (a) FCS: Representation of the confocal observation volume and movement of different fluorescent molecules: slow ones (violet) or fast (green and red). (b) Representation of the intensity traces of the moving molecules along the time in the observation volume. (c) Representation of the autocorrelation analysis (Gτ) of the fluorescence fluctuations obtained for the moving molecules. Evaluation of τD: diffusion time at the half‐maximum decay of Gτ. When corrected by the detection volume, provides the diffusion coefficient. As shown, violet molecules (slower) have higher τD, which is lower and similar for red and green molecules. The amplitude of the Gτ function is inversely proportional to the number of fluorescent molecules in the confocal volume (N). In the example, violet and green molecules are in the same concentration, while red ones are more concentrated.
Figure 5. Graphical representation of the bean emission spectrum pattern (a) and the achieved PSF (b) in confocal microscopy and SR systems based on confocal microscopy. (A) Confocal Microscopy. The airy disk pattern showing the Airy's first ring and the less intense rings that restrict the resolution of the system (a) and the obtained PSF as three‐dimensional representation of the Airy disk pattern (b). (B) STED. The airy disk pattern with the represented donought depletion beam pattern (in red) (a) and the resultant PSF with the depletion pattern beam (in red) (b) blocking the signals corresponding to the diffraction, so the obtained SR signal is restricted to the central region. (C) SR Spinning Disk. The modified Nipkow disk pattern consists of two rotating micro‐lens arrays. Here, we show the lower disk with multiple micro‐lens pinholes, which have a fixed size and are separated to avoid light diffraction (a) and the obtained restricted PSF with high resolution (b). (D) Cell Microdetectors. The concentrically arranged hexagonal detector array (a) and the obtained PSF's corresponding to the different displacements, that are reassigned by pixel to obtain a restricted calculated PSF with high resolution (b). (E) Software. The airy disk pattern of an optical system, in this case, a confocal microscopy (a) and the obtained virtual PSF calculated with an algorithm in order to extract the information with the best resolution (b).
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Gutierrez‐Erlandsson, Sylvia, Morales‐García, M Dolores, and Muñoz‐Alcalá, Angeles(Jun 2020) Confocal Microscopy. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0028799]