Currently this is a page under construction as the microscopes get setup.

The login information for both of these scopes is the same: vhlab1

Software information for PC:

Both have MATLAB 2020 with the following toolboxes:

Computer Vision

Curve Fitting Toolbox

Data Acquisition

Image Acquisition

Image Processing

Instrument Control


Parallel Computing

Signal Processing

Statistics and Machine Learning

Security Recovery Questions:

- for Scope in 308

What is your first pet's name?


What's the first name of your oldest cousin?


What's the name of the first school you attended?


- for Scope in 316:

What is your first pet's name?


What city were your parents born?


What's the name of the first school you attended?


A crash course in 2P courtesy of Joshua Trachtenberg:

Q: why not just use normal epifluorescene (shine blue light to excite a green fluorophore) to image neurons in cortex?

A: shorter wavelengths scatter more than longer wavelengths. in the cortex, where the cell bodies are lipid spheres (thus, lenses) and there's a lot of myelinated axons (thus, white, scattering tissue), the blue light just doesn't penetrate past about 50 to 100 microns. so it is pretty much useless. that said, the blue light approach has worked for mini-scopes, but only when you use a GRIN lens and only for cells right at the base of the lens. for real imaging, its useless.

Q: so what wavelength do we use?

A: all we are doing with regular fluorescence is energy transfer. a blue photon "hits" an electron (it doesn't really - its actually a constructive wave interference). that electron absorbs the energy of the blue photon (blue has more energy than red). the electron now has too much energy to stay in its current shell. so it moves to the next higher energy shell (it quantum tunnels to it). in that shell it loses a little bit of energy. this is known as a Stokes shift. it now doesn't have quite enough energy to remain in that shell, so it quantum tunnels back down to its original state. but it has to give off the energy it absorbed to do so. it already lost a little energy during the Stokes shift, so it has to give off the remainder. this is given off in the form of a new photon, but this photon has a bit less energy than the blue photon. thus, its green.

anyway, we don't really need a blue photon. we just need the energy of a blue photon. it turns out that two photons that are much lower energy can combine their energies to give the same amount of energy as one blue photon. typically this is in the form of two photons at 920nm, or in that range.

so...we just need to get one electron to be "hit" by two infra-red photons at the same time in order to get the necessary energy transfer.

it turns out that this is pretty difficult. the probability that two photons will "hit" one electron in approximately the same instant is really, really, really, really low. like, if you stood outside with a cuvette that contained a fluorophore in the middle of the day. the intensity of sunlight is only high enough to achieve a 2-photon event maybe once in the lifetime of the universe. its super improbably. the way we improve our odds is to add more photons. the more photons per unit volume, the better our chances that two of them will hit an electron at the same time. so we buy a laser that puts out a lot of photons per unit time and then we focus these photons onto a small region of space using an objective lens.

Q: won't all that infrared light at such high intensities just cook the brain?

A: yes! it will.

Q: how do we overcome that?

A: we get a pulsed laser. in a pulsed laser, photons are emitted in bursts. so sometimes there's light. somethimes theres not. in your laser, you get a burst of photons that occurs 80 million times each second. that's going to be important later. the thing is that that burst is really short. it only lasts about 100 femto seconds (that's 1x10^-13 seconds). and then its off for the next 12.5 nanoseconds. a nanosecond is 10^-9 seconds. so 12 nanoseconds is about 10 nanoseconds which is about 10^-8. we can do the math and it turns out that the laser is off a lot longer than its on. its only off for 100,000 times the duration that its on (10^-8/10^-13) = 10^5.

that's good for us. that means that the average power is 1/100,000 the peak power.

a pulsed laser lets us get to the very high light intensities needed to achieve a 2photon event, while at the same time keeping average power quite low. the cooking of the tissue is dependent on the average power while the fluorescence is dependent on the peak power!

Q: what about that 80 million times a second pulse rate? why is that important?

A: that's lecture #2