Ch_226_Final_Paper.pdf

Two-photon microscopy enables sub-diffraction limit characterizations of

millimeter-depth features in living specimens

Shawn Yoshida

1

2

1

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA

2

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA

(Dated: 5/31/2022)

Two-photon microscopy allows for the imaging of both fixed and live tissues orders of magnitude

deeper than what is possible with standard widefield microscopy techniques and does so at higher

resolutions than those conventional techniques. There are numerous structures that were previously

unable to be imaged in live specimens until the development of two-photon microscopy due to their

depth in the tissue. While confocal microscopy can also image deeper into tissues than conventional

widefield techniques, the penetration depth is still insufficient for many biological structures of

interest. Before the development of two-photon microscopy, the primary methods for imaging such

structures were tissue sectioning and scaling, i.e. making the tissues transparent via soaking in

chemical solvents. Both of these methods are incompatible with living samples, and while they

can also achieve two-photon-like resolutions, they cannot visualize any dynamics. Two-photon

microscopy exploits the nonlinearity of its namesake effect, and by only exciting near the waist of

the beam in the imaging plane, is able to achieve millimeter-depth imaging in tissues at higher

resolutions.

Keywords:

Two-photon microscopy, two-photon excitation, confocal microscopy, fluorescence,

nonlinear optics, biological imaging

I. INTRODUCTION

There are numerous cases where the spatiotemporal

organization of deep tissues and the biological processes

within them were previously not able to be studied in live

specimens. Two-photon microscopy (and later, multi-

photon derivatives) enabled the study of such structures

where no previously existing techniques could, such as

its original intended target, the neuron [1, 4], along with

more recent applications to systems like skin cells [2] and

cancer metastasis [3]. In addition, is has been applied to

the live imaging of numerous organisms such as fruit flies

[5], zebrafish [6], mice [7], and humans [2].

Many of two-photon microscopy’s benefits become ap-

parent when compared to alternative techniques. Con-

ventional widefield fluorescence microscopy techniques

are not very well suited for deep tissue imaging. While

these techniques can generally illuminate up to around

700

μm

, they cannot clearly resolve anything at that

depth, and while total internal reflection fluorescence mi-

croscopy (TIRF microscopy) is gaining popularity for its

sensitivity, it is generally limited to penetration depths of

one to two hundred nanometers [8]. Confocal microscopy

is often used for three-dimensional imaging deeper in tis-

sues, but it is limited to imaging structures up to a few

microns deep [9, 10]. Scientists interested in deeper struc-

tures like the brain were initially able to circumvent the

limit on imaging depth by euthanizing the animals, then

taking brain slices [11]. Later, scaling, a chemical method

of making a mouse embryo optically transparent enough

to directly image the brain was developed [12]. How-

ever, both of these techniques relied on the termination

of the specimen before imaging. In order to probe the

dynamics of neuronal activity, scientists needed a way

to image deep into animal tissue without the need for

euthenasia. Two-photon microscopy filled this gap, and

is able to image over a millimeter deep into tissue. In

addition, it does so at a resolution higher than conven-

tional widefield techniques, and comparable to confocal

microscopy: well below the diffraction limit. After in-

troducing the principles and practical considerations be-

hind two-photon microscopy, the underlying reason for

both increased penetration depth and resolution will be

explained.

Two-photon microscopy relies on two-photon exci-

tations (or more fundamentally, absorptions), a phe-

nomenon initially theorized by Dr.

Maria G ̈oppert-

Mayer in her doctoral dissertation [13]. As shown in

figure 1, a fluorophore that is typically is excited by a

photon with energy

hν

can also be excited by

the near simultaneous arrival (within a femtosecond) of

two photons with energies

hν

1

2

. A large

amount of energy (generally hundreds of thousands of

watts) is needed to guarantee a sufficient number of two-

photon excitations. Continuous wave lasers (CW lasers)

cannot maintain sufficient powers, so pulsed lasers like

Ti-Sapphire lasers which can produce pulses of around

150

000

at a frequencies around 80

MHz

are used in

their stead. The benefits of replacing a more simple, sta-

tistically likely excitation scheme with a less accessible

nonlinear one may not be immediately obvious. However,

the nonlinear nature of two-photon excitations is actually

responsible for both two-photon microscopy’s ability to

achieve millimeter-depth imaging and its ability to image

with increased resolution.

FIG. 1. Jab lo ́nski diagram comparing one-photon and two-

photon excitation events. For a two-photon excitation to oc-

cur, it is necessary that the combined energies of the red pho-

tons equal the energy of the blue photon, i.e.

hν

= 2

hν

and that both red photons arrive within a femtosecond of one

another. Of course, there is no requirement that the photons

actually be red or blue, the colors are just for illustrative pur-

poses.

FIG. 2. The absorption coefficients of common biological ma-

terials. While the absorption coefficients of water and lipids

increase with wavelengths in the red to infrared, the rele-

vant materials in millimeter-depth biological imaging show

decreased absorption coefficients under the same range of

wavelengths. Reproduced from [14] under Attribution 4.0 In-

ternational (CC BY 4.0).

II. TWO-PHOTON ENABLES

MILLIMETER-DEPTH IMAGING

The primary optical effects that limit imaging depth in

live tissues are scattering and absorption. Two-photon

microscopy allows us to reduce the impacts of both of

these interactions. It is often preferred that the fluo-

rophores used in biological imaging have emission wave-

lengths in the visible. Consequently, when using a two-

photon process to excite such fluorophores, an excitation

beam with a wavelength in the red to infrared regime

must be used. It is well established that the intensity of

Rayleigh scattering scales with the inverse of the wave-

length to the fourth power, i.e.

∝

1

4

. Then, it follows

that increasing the wavelength of our excitation beam

reduces the amount of scattering in the sample.

Using an excitation beam with a longer wavelength also

reduces the amount of absorption in the tissue. As shown

in figure 2, while the absorption coefficient of water and

lipids is higher at higher wavelengths, the relevant bio-

logical mass in millimeter-depth imaging like blood and

melanin feature dropoffs in their absorption coefficients

at longer wavelengths (in particular in the red to infrared

regime). Two-photon microscopy allows for the imag-

ing of standard, biological fluorophores using excitation

beam wavelengths that scatter and absorb less in living

tissues.

Of course, the penetration of the excitation beam is

not the sole factor in determining the maximum imag-

ing depth of a given technique. In order to image deeper

in the tissue while avoiding the detection of fluorescence

from areas not of interest (and not in focus), millimeter-

depth imaging techniques like two-photon microscopy

need some way to isolate the signal from the imaging

plane before detection. Because of both the rarity of si-

multaneous photon arrival and the fact that the emission

intensity scales with the square of the excitation intensity,

the only place in the beam where two-photon excitation

occurs is near the waist, where the photon flux is max-

imized. In other words, the nonlinearity of two-photon

excitations guarantees that the bulk of the detected sig-

nal originates from the imaging plane (as shown in the

bottom pane of figure 3). The size and position of the

excitation volume are also responsible for two-photon mi-

croscopy’s ability to image at a higher resolution than

conventional widefield techniques.

III. TWO-PHOTON ENABLES

SUB-DIFFRACTION LIMIT IMAGING

The resolution of light microscopes was traditionally

thought to be fundamentally limited by the diffraction

limit, given as

∗

(1)

where NA represents the numerical aperture, or the

range of angles over which a lens, in this case the ob-

jective lens, can accept light. This means that in gen-

eral, fluorescence microscopes used for biological imaging

are limited to resolutions of a few hundred nanometers.

Numerous techniques have been pioneered that circum-

vent this limit, broadly referred to as super-resolution

microscopies. While there are numerous types of super-

FIG. 3. Scheme comparing the optical excitation and detection paths of widefield, confocal, and two-photon microscopy.

Notably, confocal microscopy acheives higher resolution both laterally and axially by using a pinhole in the excitation beampath

(non shown here) to narrow the focal volume and another pinhole between the tube lens and the detector in order to block out

of focus light. Two-photon microscopy has no need for these pinholes because the nonlinear nature of two-photon excitations

constrains the excited region to only near the waist of the beam where the photon flux is maximized. The focal and excitation

volumes are depicted in the bottom pane.

resolution techniques, such as localization-based tech-

niques like STORM, PALM, and fPALM ([15–18]) and

correlation-based techniques like SOFI and variants ([19–

21]), they all require global single-molecule resolution,

a feat generally only achievable at or near interfaces.

While these techniques can achieve near-molecular res-

olutions, they are not yet suitable for the investigation of

millimeter-depth features. However, there are other ways

to get more modest increases in resolution, one of which

is by preventing out of focus (both lateral and axial) light

from reaching the detector, an effect most easily under-

stood by comparing conventional widefield and confocal

microscopes.

Traditional widefield microscopies have their resolu-

tions limited partially by the presence of out of focus light

in both the lateral and axial directions. Generally, con-

focal microscopy filters out the out of focus light in order

to achieve higher resolutions at the cost of field of view.

This is done through the use of a set of pinholes. The

first is used just before the beampath intersects with the

dichroic (colloquially known as the illumination pinhole

and not shown in figure 3 due to space) and is respon-

sible for ensuring that the light source is as close to a

point light source as possible. The second pinhole (collo-

quially known as the detection pinhole) is responsible for

filtering out all the out of focus signal that results from

excitation events outside of the imaging plane. As shown

in the bottom pane of figure 3, the entire focal volume is

excited in the confocal scheme. Much of the out of focus

signal from the focal volume can be filtered out by the

pinhole, but a combination of scattering and absorption

still limits the imaging depth of confocal microscopy.

However, due to the nonlinearity of two-photon mi-

croscopy, nearly all of the signal from the sample is guar-

anteed to be from the imaging plane. As shown in figure

3, while the excitation beam still passes through much

of the sample, only the fluorophores near the waist of

the beam (incidentally in the imaging plane) are excited.

This means that there is little to no out of focus light

to be filtered out, and two-photon microscopy is able to

achieve a finer resolution in a similar manner as confocal

microscopy without a need for pinholes. In addition, fluo-

rescent signal that is scattered by the sample is generally

filtered out by the pinholes in confocal microscopy, but

in two-photon microscopy, there is no need. Because the

signal is nearly guaranteed to be from the focal volume

at the imaging plane, scattered fluorescent signal can still

be used by the one dimensional detector, often a photo-

multiplier tube (PMT), and actually serves to increase

the sensitivity of detection in two-photon microscopy.

IV. CONCLUSION

The investigation of millimeter-depth tissues in live

specimens was long impossible due an inability of lin-

ear fluorescence microscopy techniques to resolve fea-

tures more than a few microns deep in biological tissue.

These deeper features could be imaged by cutting slices

of tissue and making specimens transparent by chem-

ical means, but neither of these methods can be per-

formed in live specimens. The nonlinearity of the two-

photon excitations that underly two-photon microscopy

enables the use of red to infrared wavelengths to specifi-

cally excite the small volume where the excitation beam

and imaging plane intersect. This permits the imaging

of live, millimeter-depth biological features (unachiev-

able by confocal microscopy) at resolutions greater than

achievable with conventional widefield techniques. It is

for this reason that the development of two-photon mi-

croscopy has had such a major impact on biology as a

whole.

ACKNOWLEDGMENTS

Thanks to Profs Geoff Blake and Scott Cushing for

a great quarter and to Danika Nimlos for TAing and

grading my (at times shocking) attempts to complete the

problem sets.

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