Epi-fluorescence microscopy

Abstract

Epi-fluorescence microscopy is available in most life sciences research laboratories, and when optimized can be a central laboratory tool. In this chapter, the epi-fluorescence light path is introduced and the various components are discussed in detail. Recommendations are made for incident lamp light sources, excitation and emission filters, dichroic mirrors, objective lenses, and charge-coupled device (CCD) cameras in order to obtain the most sensitive epi-fluorescence microscope. The even illumination of metal-halide lamps combined with new "hard" coated filters and mirrors, a high resolution monochrome CCD camera, and a high NA objective lens are all recommended for high resolution and high sensitivity fluorescence imaging. Recommendations are also made for multicolor imaging with the use of monochrome cameras, motorized filter turrets, individual filter cubes, and corresponding dyes being the best choice for sensitive, high resolution multicolor imaging. Images should be collected using Nyquist sampling and images should be corrected for background intensity contributions and nonuniform illumination across the field of view. Photostable fluorescent probes and proteins that absorb a lot of light (i.e., high extinction co-efficients) and generate a lot of fluorescence signal (i.e., high quantum yields) are optimal. A neuronal immune-fluorescence labeling protocol is also presented. Finally, in order to maximize the utility of sensitive wide-field microscopes and generate the highest resolution images with high signal-to-noise, advice for combining wide-field epi-fluorescence imaging with restorative image deconvolution is presented.

Fig. 1

(a) Cut through of an Olympus IX81 fully motorized epi-fluorescence microscope showing details of the fluorescence light path. (b) Detailed view of the epi-illumination arm for the IX81 microscope showing components of the fluorescence light path. Images (a, b) are courtesy of Michael Davidson, Florida State University, National High Magnetic Field Laboratory, Tallahassee, FL. (c) Schematic of a fluorescence cube for EGFP imaging. Blue light is selected from the white incident light from the lamp using a blue band pass excitation filter. The blue light reflects off the dichroic mirror and is directed upwards to the objective lens then focused on the sample. Green emission light from the sample passes through the dichroic mirror and through a green band pass emission filter to the detector. The emission filter also reduces image background by rejecting any reflected blue incident light.

Fig. 2

(a) Filter transmission curves for EGFP cubes using either standard soft coated filters (HQ series) or hard coated filters (ET series) from Chroma Technology. (b, c) Cells stained with phalloidin AlexaFluor® fluorophore 488 (Invitrogen) were imaged on a Zeiss Axiovert 200 M microscope with a 63×/1.4 NA oil immersion lens and Axiocam HR camera at full resolution with 500 ms exposure times. Images were taken with either (b) the Zeiss soft coated 13 FITC; or the (c) Chroma Technology ET-GFP 49002 filter cube sets. The mercury lamp was attenuated to 5% power with neutral density filters. The image contrast, brightness, and gamma factor were all adjusted to the same levels for images displayed in (b, c). (d) Intensity pro file from the white line shown in (b, c) for the hard coated ET (solid line) and soft coated HQ (dashed and dotted line) filter sets. The gamma factor was used to bring up dim features within the images so that they are visible by eye. Scale bar is 10 μm.

Fig. 3

Images of BPAE cell slide from Molecular Expressions labeled with DAPI, AlexaFluor® 488 Phalloidin, and MitoTracker® CMXRos. Images were collected on an Olympus IX71 microscope coupled with a mercury HBO lamp and using DAPI, FITC, and Texas Red BrightLine® hard coated filter sets (Semrock). Images were collected on a Retiga 2000R camera at full resolution with the same settings using three different 20× lenses. The lamp intensity was attenuated to 6%, with exposure times of 250 ms for DAPI, 1,000 ms for AlexaFluor® 488, and 500 ms for MitoTracker® used. Lenses used were (a) Plan 20×/0.4 NA; (b) UPlanFL N 20×/0.5NA; (c) UPlanSApo 20×/0.75NA. Images of the same sample as in (a–c) were collected using a 40×/0.6NA LWD, U PLAN FLlens with a coverslip thickness correction collar. Images were collected with the collar set correctly to 0.17 mm (d) or incorrectly to 1 mm (e). The image contrast, brightness, and gamma factor were all adjusted to the same levels for the images displayed in (a–c) as well as and for (d, e). The gamma factor was used to bring up dim features so they are visible within the images by eye. Scale bar is 10 μm.

Fig. 4

Images of the same sample collected on the same microscope as in Fig. 2. Images were captured with a Retiga 2000R monochrome (a, c) or color (b, d) camera at full resolution with a 60×/1.42 NA, PlanApoN objective. All images were collected with the same settings with the lamp intensity attenuated to 6% and exposures of 200 ms for DAPI, 500 ms for AlexaFluor® 488, and 50 ms for MitoTracker®. Images in (a, b) are presented with the same image display settings. The gamma factor is used to bring up dim features so they are visible within the images. Images in (c, d) are zoomed in areas of the images in (a, b). Images in (d) were further adjusted for brightness and contrast so they appear to have similar brightness when compared to (c) in order to provide a fair comparison of the image resolution. Scale bars are 10 μm.

Fig. 5

Neurons were fixed with 4% paraformaldehyde/4% sucrose in phosphate buffered saline (PBS) for 15 min at room temperature and permeabilized with 0.2% Triton X-100 for 5 min at room temperature. (a) Cells were stained with a SV2 mouse monoclonal antibody (1:250 dilution) from the Developmental Studies Hybridoma Bank (The University of Iowa, Iowa City, IA) and anti-mouse Alexa Fluor® 488 (1:500 dilution). (b) Cells were co-stained with phalloidin-TRITC (1:250 dilution) from Sigma (St. Louis, MO) by incubating for 1 h at room temperature in the same step as the primary SV2 antibody. Neurons were imaged with a Retiga EXi CCD camera (QImaging, Surrey, BC) attached to an Olympus IX71 inverted microscope (Melville, NY) with a 60×, PlanApo/1.45 NA objective (Olympus). Image acquisition was controlled by MetaMorph software (Molecular Devices, Sunnyvale, CA) interfaced with a Lambda 10-2 automated controller (Sutter Instruments, Novato, CA). For SV2, an Endow GFP Band pass filter cube (excitation HQ470/40, emission HQ525/50, Q495LP dichroic mirror) (Chroma, Brattleboro, VT) was used. Phalloidin was imaged with a Chroma TRITC/Cy3 cube (excitation HQ545/30, emission HQ610/75, Q570LP dichroic mirror). Scale Bar is 10 μm.

Fig. 6

Images of living CHO-K1 cells stably expressing paxillin-EGFP were collected with a CoolSNAP EZ camera with 2 × 2 binning on an Olympus IX71 microscope equipped with a 60×/1.45 NA oil immersion objective. Excitation was from a mercury lamp using a custom EGFP cube from Chroma Technologies (hq480/20×, z488rdc, hq525/50 m). (a) Image corrected for the background intensity of an ROI. (b) Image of the background intensity in a region of the sample with no cells collected with the same parameters as in (a). (c) Image corrected using a normalized background intensity image of (b), followed by subtraction of the average background intensity of an ROI with no cells. The brightness, contrast, and gamma factor are set to the same levels for (a, c), but were adjusted in order to visualize the background intensity in (b). Scale bar is 10 μm.

Fig. 7

Images of (a) DAPI (Zeiss 49 DAPI cube; 250 ms), (b) phalloidin AlexaFluor® 488 (Zeiss 13 FITC cube; 400 ms), and (c) MitoTracker® Red CMXRos (Chroma Technology ET-Texas Red cube, 600 ms) taken on the same microscope with the same camera settings as in Fig. 2. The mercury lamp was attenuated to 5% power with neutral density filters. Z-axis images were taken every 0.1 μm for a total of 82 image frames. (a–c) Images are maximum projections of nine images centered about the focal plane for the raw widefield images (first column), and images deconvolved with AutoQuant X (third column). The second and third columns are displayed with the same brightness, contrast, and gamma factor settings to show the increase in S/N with deconvolution. The gamma factor was used to bring up dim features so they are visible in the images. The first column is displayed with enhanced brightness and contrast so that image resolution can be visualized. Scale bar is 10 μm.

Fig. 8

A color overlay of the maximum projection of the same data from Fig. 7 with blue (DAPI), green (actin phalloidin AlexaFluor® 488), and red (MitoTracker® Red) using AutoQuant X (Media Cybernetics) software for the (a) raw data and (c) deconvolved data. (a, c) also show x-z (bottom) and y-z (right side) axial profiles through the image stack emphasizing reduction in out-of-focus light following deconvolution. The same images were processed with Imaris 6.1.5 software (Bitplane Inc.) and 3D iso-surfaces were automatically produced from the (b) raw and (c) deconvolved images. Scale bar is 10 μm.