Quiz #4 (Part #1, Live Cell Imaging)

Spinning Disk Confocality

Is typically confocal for high NA objectives (High Numerical Aperture)

Concept from Confocal Microscopy (like LSCM)

Uses a detector pinhole to reject stray light from above or below the focal plane,

The Triangle of Compromise

Live cell imaging involves this triangle. Three points of this compromise are Resolution, Speed, and sensitivity. 

Purpose of the triangle of compromise 

Maximize all three. Resolution, Speed , and Sensitivity.

Basic Requirements for Live Cell Imaging (5)

1) Labeling your sample.

2) Incubation

3) Focus maintenance

4) Signal detection (PMTs or cameras)

5) Gentle imaging

How did we image living cells?

You can image living cells under bright-field, or phase, DIC etc.

What the limit to Bright-field?

Difficult to image organelles or other sub-cellular details 

Labeling Your Cells (Sample) 

• with Fluorescent Markers: are typically very specific and give high contrast ex) Mito/ER tracker, Organelle Lights, Tubulin tracker, etc.

• Fluorescent Proteins: can also be very specific, but may or may not give high contrast, ex) GFP and variants 

• Optical Highlighter Probes: Are photoactivateable or photoswitchable probes 

What is the Goal of Incubation? (Req #2)

The ultimate goal is to keep your cells happy. Considerations: when incubating (if necessary) , you must control pH, temperature, and humidity

Focus Maintenance (Req #3)

Focus maintenance corrects for thermal drift over time or other changes in focus.

What is thermal drift?

Thermal drift is the slow, unwanted movement or shift in focus, position, or alignment of a microscope’s components over time, caused by tiny changes in temperature.

Two way of focus maintenance? 

• Software based: Is one possible way, but it is difficult and not as reliable to implement. 

• Hardware-based (Preferred): Different companies have proprietary systems: 

- Nikon: Perfect focus 

- Zeiss: Definitive focus 

- Leica: Adaptive focus 

- Olympus: Zero drift 

How do we improve things in terms of Media?

Immersion media (oil,water,glycerin, silicone,etc.)

Oil immersion

Most objectives in the 60x to 100x (and higher) magnification range are designed for use with immersion oil

Optimal Refractive Index (RI)

Good results are obtained with oil having an RI (n) of 1.51. This RI is precisely matched to the refractive index of glass. Using immersion oil prevents light loss due to refraction or internal reflections that occur when using air. 

Signal Detection (PMT’s or Cameras), Req #4

Detection Devices: Signal detection is done with PMT’s (Photomultiplier Tubes) or cameras.

Quantum Efficiency (QE), Resolution, and Speed

• QE: Typically low for PMT’s and higher for cameras

• Resolution: Typically higher for PMT’s and lower for cameras

• Speed: A key consideration is how fast you need to require images.

Bit more on detection 

• Nyquist Sampling Principle: the digitizing device must use a sampling interval that is no greater than one-half the size of the smallest resolvable feature of the optical image. 

• Practical Rule: To capture the smallest detail, sampling must occur at a rate where a minimum of two samples are collected for each feature. 

• Resolution (in X,Y) = λ/2NA

• Resolution (in Z) = 2λη/(NA)2

• Nyquist also applies time 

Gentle Imaging (Req #5)

• Goal: To prevent damage or abnormal cell behavior during long-term imaging.

• Factors to Minimize: Laser power, mercury lamp power, exposure time, scan size, etc..

• Concern: Reducing light exposure helps minimize photobleaching.

Experiment that day

• Planned Experiment: Visualizing yeast FIM-GFP (endocytic patches) and PPC89-mCh (spindle pole body).

• Related Fields: Mentions microfluidics in developmental biology and studies on physical forces affecting bacteria's toxin resistance and antibiotic pumping.

• Result: The goal is to obtain live cell movies.