Introduction to Fluorescence Microscopy

Introduction to fluorescence microscopy Fluorescence Widefield Fluorescence microscopes Filters and Dichroics Objectives and abberation CCD cameras

Natural Fluorescence

Transmittance is subtractive while fluorescence is additive

excitation emission Fluorescence Microscopy: basics of theory Absorbance spectrum limits excitation. Energy states limit excitation Molecule returns to lowest vibrational state emitting heat Light is emitted on return to ground state

Fluorescence spectra correlated to sise of conjugated ring

Multichannel fluorescence labelling Direct coupling to macromolecules Fluorescent dyes and substrates Fluorescent fusion proteins Fluorescent Antibodies Ch2(Red) Texas Red anti-rabbit & Rabbit anti-  Gal Ch1(Green) UBI-GFP Arterial edothelial cell Ch1(Green) FITC Tubulin Ch2(Red) mitotracker Ch3(Blue) DAPI Ch1 Ch2

Light path of a Epi-fluorescent Microscope fluoromicroanatomy.html

Upright Scope Brightfield Source Epi- illumination Source Image from Nikon promotional materials

Inverted Microscope Brightfield Source Epi- illumination Source Image from Nikon promotional materials

Microscope for widefield epifluorescence Mercury Lamp Dichroic Reflector Objective Sample Stage DIC analyser Dichroic Reflector CCD camera

Mercury lamp spectra

Standard Band Pass Filters Transmitted LightWhite Light Source 625/50 nm BandPass Filter Texas Red nm Light

Standard Long Pass Filters Transmitted Light Light Source 500 nm Long Pass Filter >520 nm Light Transmitted Light Light Source 575 nm Short Pass Filter <575 nm Light Standard Short Pass Filters

Optical Filters Dichroic Filter/Mirror at 45 deg Reflected light Transmitted LightLight Source 510 LP dichroic Mirror

GFP Longpass Emission Set D425/60x 470DCXR E480LP

Dichroic for multi-colour samples The excitation (red/oarnge) and emission (blue) spectra for three common fluors (Alexa 488, 555 and 633) Requires a dichroic mirror with three bands of reflection and intervening windows of transmission

Dielectric filter components “glue” Interference in Thin Films Small amounts of incident light are reflected at the interface between two material of different RI Thickness of the material will alter the constructive or destructive interference patterns - increasing or decreasing certain wavelengths Optical filters can thus be created that “interfere” with the normal transmission of light

Microscope for widefield epifluorescence Mercury Lamp Dichroic Reflector Objective Sample Stage DIC analyser Dichroic Reflector CCD camera

The objective determines the content of your image! The objective is critical to the efficiency of light collection (your signal) And determines the accuracy of the image (inaccuracy is called aberration). The objective determines resolution.

Objective markings PLAN-APO-40X 1.30 N.A. Oil 160/0.22 Flat field Apochromat Magnification Numerical Tube Coverglass Factor coloured rings Aperture Length Thickness  - Infinity corrected Immersion medium Oil (black ring) Water (W) Air (white ring)

A  NA=n(sin  ) Light cone (n=refractive index) Resolving power is directly related to numerical aperture. The higher the NA the greater the resolution Resolving power: The ability of an objective to resolve two distinct lines very close together NA = n sin  –  is 1/2 the angular aperture of the objective Numerical Aperture

Limits of resolution result from interference Rayleigh limit for self-luminous objects Abbé limit for illuminated objecs

Microscope Objectives Images from

Microscope Objectives Specimen Coverslip Oil Microscope Objective Stage 60x 1.4 NA PlanApo Refractive Index Cells Water glycerol Glass 1.52 Zeiss Oil Diamond 2.42

Refractive Index Objective n=1.52 Specimen Coverslip Oil n=1.33 n = 1.52 n = 1.0 n = 1.5 Water n=1.52 Air

Spherical Aberration Peripheral ray focus is shorter than more central (paraxial) ray focus. Compromise is ‘Circle of Least Confusion’

Monochromatic Aberrations - Coma Coma is when a streaking radial distortion occurs for object points away from the optical axis. It should be noted that most coma is experienced “off axis” and therefore, should be less of a problem in confocal systems Images reproduced from:

Monochromatic Aberrations - Astigmatism If a perfectly symmetrical image field is moved off axis, it becomes either radially or tangentially elongated. Images reproduced from:

Microscope for widefield epifluorescence Mercury Lamp Dichroic Reflector Objective Sample Stage DIC analyser Dichroic Reflector CCD camera

The CCD Camera Extended red sensitivity 1344(H) X1024(V) pixels 8.3 frames per second (to 45 fps with 8X8 bin)

Scanning the CCD chip

Binning 2X2 16fps 4X4 28fps 8x8 45fps Full well capacity (18000 electrons) Bloom Readout noise (6 electrons) Dynamic Range (6/18000 = 3000)

Electron Multiplying Charge Coupled Device (EMCCD) Impact ionization leads to secondary electrons and amplification before readout Amplification can be as much as 1000x Readout noise is excluded but shot noise is amplified

Fluorescence Microscopy in the Sussex Centre for Advanced Microscopy Roger Phillips Biols 2C9,10,11 Ext 7585