Key Concepts in Optical Imaging Part III: Fluorescence Techniques

Aaron Taylor, PhD Managing Director BRCF Microscopy Core [email protected] Fluorescence-Based Techniques Why Fluorescence? Fluorescent dyes are useful because the intensity of emissions can be quantified (unlike colorimetric stains) and sensitivity is increases since ‘background’ is dark (unlike for transmitted light).

Exciting Light FITC

GFP ‘Energy levels’

Fluorescence Electrons

-Lasts 1-10 nanoseconds -Longer wavelength -Highly Inefficient (~10-6) Nucleus -Brightness = Quantum Yield x Extinction Coefficient Finding the Spectra for Your Dyes

Various databases show the excitation and emission spectra for virtually all fluorophores. Review of Bright-field light path

Image Kohler Illumination Formation Visualization

Sample

Lamp Interm. Perceived Image Eye Image Collector Condenser Objective Eye Tube Piece Lens Lens Lenses Lens

Because fluorescence is super inefficient (~10-6), the excitation light can not travel towards the objective. There is no filter good enough to separate the emissions from the much stronger excitation. Epifluorescence Microscopy How Epifluorescence Microscopy Works

1. Excitation light is shined through (not Broad-band light source into) the objective. Collector Lens 2. The objective functions also as the condenser lens. Aperture Diaphragm

3. Color-selective filters distinguish the ‘Condenser’ Lens excitation from emissions. Field Diaphragm

Field Lens Collimated Illumination

Sample Filter ‘Cube’ Interm. Objective Lens AND Obj BFP Tube Image Condenser Lens Lens Out of Focus Emissions are a Problem

Fluorescence generated outside the focal plane still also reaches the camera, which adds ‘blur’ that can greatly reduce image contrast and sensitivity.

Light from Mercury Lamp Thick Fluorescent Camera Sample Obj BFP

Filter ‘Cube’ Interm. Objective Tube Image Lenses Lens Plane

Lots of out-of-focus light reaches the camera. Typical Epifluorescence Image

Epifluorescence Confocal Microscopy The goal of confocal microscopy

Instead of exciting the entire sample at onve with collimated light, confocal microscopy scans a point of light back-and-forth across the sample to create an image sequentially. This design allows out-of-focus light to be blocked with a small (10’s um) aperture called a pinhole.

Excitation concentration highest in focal plane Remaining out-of-plane emissions blocked by pin-hole

PMT

Objective Tube Lenses Lens

Focal Pin-hole Plane

Optical section thickness is distinct from depth of field Optical Sectioning from a Wave Perspective

Recall that due to diffraction, light is focused to an intensity profile called an Airy disk. The width of the Airy disk is measured in ‘Airy Units’. The pinhole should be ‘just big enough’ to let most of the Airy disk pass.

Note that the Airy Units in the image plane are wavelength and magnification dependent. 2D PSF 1D PSF

Intensity Intensity

PMT

Pin-hole 1 AU 2 AU Pin-hole Pin-hole How the scanning works

Mirrors attached to electric motors scan the excitation light. Feed back from the mirrors tells a computer where in the sample the point is located.

Raster Pattern

mirror (slow) mirror

- Y

X-mirror (fast) The scanning often determines resolution

Because the image is collected via scanning, sampling rate usually limits resolution in practice. Higher resolution can be obtained by A) decreasing the scan area or B) scanning more pixels…. as long as the point-to-point displacement remains greater than the width of the excitation PSF. Large Scan Area; Lowe Resolution Small Scan Area; Higher Resolution

The spot size is constant and determined by the objective’s numerical aperture. The confocal light path

A modular set of optics called the ‘scan head’ handle the point scanning, filters the excitation / emissions, and contains the pinhole and detectors.

Scan Mirrors

Scan Lens Scan head

Intermediate Laser Source image plane Dichroic Mirror

Filter

PMT Pinhole in Image Detector Plane Z-Stacks By successively moving the axial (z) location of the focal plane within the sample, a series, or ‘stack’, of (2D) optical sections can be collected that together constitute a sampled 3D image.

Move objective in z

Thick Fluorescent Sample A ‘3D’ Image Other confocal designs: Spinning-disk confocal

Many points can be scanned in parallel with the use of a spinning disk of aligned lenses and pinholes. Scan Pattern The holes are imaged onto the sample. 100s fps for one color. Pin-hole Focusing Disk Disk Dichroic Rotation of Mirror spiral hole pattern sweeps points across the scan area. Laser Source Fluorescent Sample Objective Lenses

Camera Spinning-disk confocal A confocal optical section vs epifluorescence

Confocal Epifluorescence When is confocal preferred over wide-field?

Epifluorescence Confocal

Resolution: -Diffraction limited - Diffraction limited (usually)

Light Path: -Wide-field -Point scanning

3D images?: -No - Yes - Sort of with deconv.

Sensitivity: -High (single molecule) -Low (100s molecules)

Speed: - High (20 msec) - Slow (+1 sec)

Photodamage: - ‘1x’ per image - ‘10-100x’ per image Multiphoton Microscopy Goal of Multiphoton

Confocal can only image about 100 um into most samples. Beyond this, too much light is lost due to absorption and scatter by the sample. Multiphoton uses pulsed infrared excitation light and specially placed detectors to reduce and work with scattered light.

Intensity Tissue

–Єc D Depth T = e

100 um NIR light penetrates tissue ~100x better than visible wavelengths

From: Weissleder. Nat Biotech. 2001 The Multiphoton Effect Multiphoton (aka ‘two-photon’) is based on a physical process called two-photon absorption where two photons interact with an electron simultaneously to cause fluorescence. Thus, each photon needs only half the energy relative to single photon absorption.

Multiphoton Fluorescence Emitted Light: ~530 nm Exciting Light: ~980 nm

Standard Fluorescence Exciting Light: ~490 nm Electrons ‘Energy levels’ Emitted Light: ~530 nm

Nucleus Multiphoton is point scanning, just like confocal

Multiphoton light path is same as for confocal, except detectors usually sit just behind objective or on sample, not behind a pinhole.

Scan Mirrors

Scan Lens Scan head Intermediate Laser Source Dichroic image plane Mirror

Filter

No Pinhole Point Detector (PMT) Multiphoton inherently optical sections

Multiphoton effect is very rare (like lighting striking twice!) so it only really happens where the excitation light is most concentrated at the focal plane. Thus, it increases as the square of the incidence power.

Focused NIR excitation light Fluorescence only generated here.

Focal Plane Scattered light can be used to image

Since excitation is know to happen only in the image plane, any emissions that are collected can be used for imaging. The emissions need not follow straight ray paths or even enter the objective!

Scattered emissions

Focal Plane

Scanning Electron Microscopy SEM is also a point scanning technique

Remember electrons are charged particles. Thus, a magnetic field can be used for focusing and also for point scanning.

Ejected (SE) or scattered (BSE) electrons are detected. Light Sheet Microscopy Why not confocal?

Biologists often want to image large 3D volumes, but:…

1. Point scanning techniques are really slow. E.g. 10 us/px x 1M px/slice x 100 slices = 17 min per 100 slices.

2. Pont scanning techniques are harsh. High laser powers traveling through the entire sample for each image kills the sample and bleaches fluorophores.

Obj BFP Scan Mirrors

Point Scanning Confocal Fluorescent Sample Objective Lens What is light sheet microscopy?

Light sheet microscopes use one light path for excitation and a separate light path for detection. The excitation path creates a sheet of light in the sample that is perpendicular to the detection path axis:

Camera

Objective

Emitted Light Excitation Light Thick Sample

Sample is moved or sheet is scanned to collect stack. Many different kinds of light sheets! Longitudinal Type BFP Lens Cross-Section Thickness

Cylindrical Low All Optical LS Lens NA >2 um (Scanning not required)

Low Scanned LS NA >2 um long

Scanned LS High 1 um NA short

High Bessel Beam LS NA 0.5 um

High Lattice LS NA 0.4 um

Different kinds of light sheets are used for different kinds samples! Light Sheet: Summary

1) Optical sectioning is provided by plane illumination (1 um thick) and the excitation intensity is independent of imaging depth.

2) Imaging is highly parallel (wide-field) and so is very sensitive and high speed.

3) Requires an optically ‘clear’ sample to maintain the plane illumination.

4) Huge datasets (>100 GB) are difficult to analyze. Super Resolution Microscopy Review of resolution

Recall that resolution is related to the diameter of the Airy disk, and is at best about 200 nm for a high NA oil objective.

Intensity Sum Intensity

Intensity

Sum Lens “Resolved”

Intensity “Not Sum Resolved” Lens Super Resolution Light Microscopy Super resolution microscopy is any technique that offers resolution better than the traditional 200 nm diffraction limit. All existing technologies are fluorescence-based. 1) Stochastic Optical Reconstruction Microscopy Diffraction-limited (STORM) and Photoactivated Localization Microscopy

(PALM). 200 nm 200 nm + + 2) Structured Illumination Microscopy (SIM)

200 nm

200 nm 3) Stimulated Emission Depletion Microscopy (STED)

Fine Details in 200 nm Sample Localization Microscopy STORM (or PALM) Concept Using special dyes and lasers, it is possible to image only a few dye molecules per frame. Then, image processing can be used to estimate the location of the peak of each molecule’s image, which effectively ‘throws away’ the spread of the Airy disk. After thousands of frames, a peak localization plot (the ‘image’) can be constructed. Wide-field:

Image of single molecule Estimate of peak location Intensity

Object Molecule Images Peak Location Localization Plot Many, many Localizations STORM (or PALM) is performed in TIRF mode Because STORM (PALM) requires single molecule imaging, no out-of- focus light can be tolerated – the background will obscure the signal. To create an extremely thin (50 nm) optical section, these techniques are preformed in total internal reflection fluorescence (TIRF) mode.

Wide-field Mode TIRF Mode

Aqueous Sample; RI = 1.3 sample ffp Evanescent 200 nm Field

Critical Angle objective Glass Coverslip; RI = 1.5 bfp excitation light LP is certainly NOT the resolution

Localization density is much more limiting in practice.

(5 nm pixels) (10 nm LP)

E.g. with 75% of localizations recorded, resolution is around 60 nm, given 20 nm LP. Implications

▪ Resolution of objects is rarely better than 50 nm and usually >=75 nm.

▪ Localization of molecule positions typically 20 nm.

▪ Localization microscopy is most powerful for applications where the goal is to localize individual molecules, not to resolve the size/shape of ‘objects’ comprised of many molecules.

Keep these points in mind as we talk about analysis techniques Particle averaging When all the objects are known in advance to be the same, averaging together the image of many particles can produce a higher resolution image.

(Laine, Nat Comm, 2015) Structured Illumination Microscopy SIM Uses Heterodyning

Two spots of excitation light are placed at a high NA position within the back-aperture (and usually a 3rd in the middle). Thus, a sinusoidal interference pattern of excitation light is produced in the front focal plane.

Sample

ffp sample

Coverslip objective

bfp

excitation light Moire Fringes: A 2D SIM analogy Stimulated Emission Depletion Microscopy Stimulated Emission Depletion Microscopy (STED)

STED is point scanning technique that uses an intensity annulus of light to deplete fluorescence from the edges of an Airy disk. Thus, fluorescence is generated only where depletion intensity is low, effectively narrowing the region where fluorescence occurs.

Airy disk of excitation light Annulus of depletion light superposed

Fluorescence Fluorescence generated generated only everywhere where there is no depletion

200 nm <50 nm

(Depletion emissions are removed with a filter)