Happy 2024! I hope your year is full of peace and projects. And of course, lots of blogs! 

Maybe some art too (Fig. 1). 

On my last post, I described how I plan to observe the orientation of cellulose in the root epidermis. To recap, I need to stain cellulose with a fluorescent dye and image the polarized fluorescence from the dye through a confocal fluorescence microscope. I’ll be able to do this thanks to an  ingenious system developed by Rudolf Oldenboug and team at Marine Biological Laboratory (Woods Hole, Massachusetts). Their system comprises liquid-crystal based hardware and driving software, written for the Micromanager platform. Although most of their systems are for characterizing birefringence or fluorescence in a wide-field microscope, they have one for confocal, designed around a Zeiss LSM 780, which is available at MBL; I brought their system here because the University of Birmingham’s Medical Imaging Suite has the very same model. 

Recently, I have encountered difficulties with LSM 780 here, also described in my previous post. Soon after getting off the boat (OK, off the plane), I had encountered difficulties in staining cellulose with fast scarlet, the dye I expect to use. In contrast to the balky fast scarlet, I found cooperative staining from another cellulose-binding dye, Calcofluor white. Alas, this dye is excited by ultraviolet light and short wavelength blue; the LSM 780 has a 405 nm laser, it excites the fluorescence from Calcofluor beautifully, but the 405 nm laser light might fry the liquid crystals. Probably was the verdict of the engineer I asked at the manufacturer. 

Trouble with the microscope; trouble with the dyes; what is to be done? Casting around, I discovered that the Medical Imaging Suite has a multi-photon fluorescence microscope. This high-tech microscope, like the confocal kind, was designed to reject out-of-focus light. Out-of-focus fluorescence—that is, light from dye molecules above and below the focal plane—limits conventional (aka wide-field) fluorescence microscopy. The incoming (i.e., incident) light forms a bi-conical volume spanning the thickness of sample and excites fluorescence throughout. The plane that is in focus will thus be surrounded by a pea-soup fog of fluorescence that is out of focus. This is why wide-field fluorescence works well on thin samples (and by thin I mean 10 µm thick, such as animal cells grown in culture) and rather poorly on anything thicker. 

OK, returning to confocal versus multi-photon, the former excludes the out-of-focus light by collecting light that passes through a pinhole placed at a conjugate plane¹ as the desired plane in the sample (Fig. 2). That is, light that is in focus at some depth at some point in the sample is also in focus at that pinhole and thus passes thru to the detector. But out-focus-light falls around the pinhole and is blocked. Of  course, this is light for one point in the sample focal plane; by using a laser-scanning system, an image is built up from an array of scanned points. There are tricky variations on this concept, using spinning disks or slits, but they are all basically analogous. Confocal systems work well and are in use everywhere fluorescence microscopy is happening. 

But the confocal approach has limits. As light goes deeper into a sample, it is scattered. Practically speaking, confocals can image down to ~100 µm. An order of magnitude better than wide-field but people want to go even deeper, particularly those who are studying intact structures (tissues, embryos, whole organisms). Enter the muti-photon (which I will consider in its simplest guise—the two photon) microscope. Fiendishly clever, this instrument is based on a prediction made by theoretical physicist Maria Goeppert-Meyer in her 1931 dissertation and converted to a practical technique in the 90’s and oughts. 

Living up to its name, the two-photon approach requires the dye to absorb two photons, whose energy collectively add up to enough for subsequent fluorescence (Fig. 2). As an example, Calcofluor white will fluoresce after it has absorbed a photon with a 350 nm wavelength. But energy is what matters: the dye will fluoresce after it absorbs two photons of 700 nm, each of which has half the energy of a 350 nm photon. Crucially, with just one 700 nm photon absorbed, there is no fluorescence. Now, it turns out (and this is the bit where being a theoretical physicist helps!) the chance of a dye absorbing two photons is way less than the chance of absorbing just one. To put this another way, the two-photon requirement makes the fluorescence depend strongly on the intensity. In the microscope, at the point of focus, the intensity is high (that is one way to think about what the word focus means). By contrast, intensity is lower in out-of-focus regions. Because of the non-linearity of two-photon absorption, these out-of-focus regions emit little fluorescence. As with confocal, a laser-scanning set-up is used to build an image by scanning across the field. 

Two-photon is not just a party trick. The exciting light is near infrared (commonly 800 to 1200 nm) and thus not as strongly scattered as visible wavelengths. Indeed, two-photon microscopes can image as deep as one millimeter, gaining an order of magnitude on the confocal. To be sure, with the infrared wavelengths, there is less resolution; but this is made up for by the greater depth accessible. 

With the two-photon instrument, I should be able to use ol’ reliable Calcofluor to study cellulose orientation. Further tantalizing me, the microscope part of the two-photon set-up is an upright stand and the objectives are long-working distance water immersion lenses. This means that I can image the roots while they remain undisturbed (I was going to write happy) in their agar medium. 

This holiday week just past, I did the simple thing of preparing agar medium containing 0.001% Calcofluor, which I have previously determined does not detectably alter root growth rate, and imaging roots while they remained undisturbed in the agar. For this test, I used a wide-field fluorescence microscope. Lo and behold! The agar was stained weakly by the dye but the fluorescence at the root surface was bright and clear. Next week I will see what it looks like on the two-photon microscope. 1. This is a concept from the optics of microscopy. The sensor (film or chip) of a camera, or indeed your retina, is positioned at a conjugate plane, meaning that points brought to a focus by the objective lens are also in focus. There are many such planes in a microscope; if you could put a sensor at one of them, you would record the image.