A confocal laser scanning microscope must be modified and equipped with a specialized near-infrared pulsed laser system in order to perform multiphoton imaging. The following characteristics should be used as a guide when comparing multiphoton excitation with conventional laser scanning confocal microscopy.

Multiphoton microscopywiki

You can’t randomly pick a lens and expect it to perfectly match your applications, and there are some key factors you should take into consideration. You can also refer to our guide on how to choose a lens for the Raspberry Pi High Quality Camera.

The C/CS-Mount lenses look quite similar with the same thread size. However, the C-Mount lenses are designed with a longer back focal length than the CS-Mount, so we have a C-CS adapter for the Raspberry Pi High Quality Camera to use with C-Mount lenses.

Two photon excitationmicroscopy

Another thing to mention is the aperture, namely the hole on the lens to pass light through. If you want a deeper depth of field (DoF), you’d better get a lens with an aperture ring, since a decreased aperture will increase the DoF. If you have nothing to say about the DoF or you usually shoot from a further distance, a fixed aperture will be okay for your application.

Multiphoton microscopyprocedure

Joseph R. Lakowicz - Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of Maryland and University of Maryland Biotechnology Institute (UMBI), 725 West Lombard Street, Baltimore, Maryland 21201.

Using probes such as calcium ion indicators, some of which are excited by ultraviolet light, considerations for the aberration and transmittance of ultraviolet light through the microscope optics can be eliminated in multiphoton excitation microscopy. Specimen damage from the ultraviolet light can also be reduced. Multiphoton excitation is expected strongly to photo-activate caged compounds only at a three-dimensionally specific point in specimens.

However, the new change brings cost. You must spend extra money on the lens, and more time getting to know the lens. Simple as the old Pi cameras were, they saved most people from thinking about lens, which is not included in the Raspberry Pi High Quality Camera. But how would it work without a lens?

Multiphoton microscopyprinciple

The CGL lenses recommended are at a reasonable price for the Raspberry Pi community, with the 6mm CS-Mount lens at $25 and the 16mm C-Mount lens at $50. We’ve purchased both, and admittedly, those are not bad. However, the issue is that we know little about the lenses just like this mysterious company – there is no detailed datasheet or specifications in the box, no other lenses available than the two, and even no way to contact them directly.

If you are using a CS-Mount lens, the adapter must be removed. However, for C-Mount lenses, the adapter is a must-have. Sometimes if you are still not able to focus a C-Mount camera even after the adapter is on, adjust the back focus. The gear-like ring on the main housing of the camera module is the back-focus adjustment ring, and you can spin it to alter the distance between the bottom of the lens and the IMX477 image sensor.

Haruo Kasai Laboratory - The Kasai laboratory has developed new approaches, based on two-photon excitation microscopy, to the study of neurons and secretory cells. The application of these approaches has provided important insight into the structure-function relations of central synapses and the mechanisms of exocytosis in both neurons and secretory glands such as pancreatic islets and acini.

Firstly, it’s the focal length. The focal length is the most concerning issue for most users to decide how wide the image frame you want to cover. The officially endorsed 6mm lens is usually advertised as wide angle, but it’s just slightly larger than the stock lens of V2, with a horizontal field of view at around 65 degrees. It’s the wide angle to be compared to the 16mm telephoto lens, but not the wide angle you might really need. You can refer to our focal length calculator page for more details.

Multiphotonfluorescencemicroscopy

Lens is a crucial part in a camera system that needs flexibility. A single lens cannot meet all needs because the use case varies from one application to another. Sadly, V1 and V2 Pi cameras have glued stock lenses, which leave us little to customize. Take the Raspberry Pi Camera Module V2 for example, we have to swap the whole lens-sensor unit for Arducam IMX219 drop-in replacements to mount a different lens.

Two-photonmicroscopyneuroscience

Recently, the Raspberry Pi High Quality Camera has changed this situation by officially introducing the C/CS-Mount to Pi cameras. With a standard lens mount, this new HQ camera has access to much more lens options.

Arducam has been building customized Pi camera boards with interchangeable lenses as early as the V1 era, and we offer a wide arrange of lenses other than the officially endorsed 6mm and 16mm lenses. These lenses are also compatible with Arducam IMX477 High Quality Camera Modules.

While you are adjusting the lens, we recommend you put the camera module on a tripod. As the camera module now accommodates a much heavier lens, a tripod is handy for holding the camera steady. You can also check the official guide for more information.

On the contrary, Arducam offers many lenses tested on the IMX477 in M12, CS and C Mount, ranging from telephoto to fisheye lenses. Not only do we have datasheet for the lenses, tutorials are also available on how to select the right one for you.

If you are really considering a wide-angle application, usually with an HFoV greater than 100 degrees, consider other options including the C-Mount Zoom lenses and M12 Lenses from Arducam. The zoom lens is helpful if you need to switch between a wider view and a narrower view from time to time, and the M12 lenses can push the field to the fisheye level.

Multiphoton microscopyreview

Once you’ve mounted the lens on, you will have to face the various kinds of handles on the ring of the lens. Typically, every C/CS-Mount comes with a focus ring, and many come with an aperture ring, while few may include a zoom ring. The trick to adjusting theses rings is you only tweak one ring at a time with the others fixed. Otherwise, they will move each other and ruin your last adjustment.

If you need help with the Arducam products you’ve purchased, please include the following questions in your post and answer them to help us better understand your needs.

Multiphoton microscopyvs confocalmicroscopy

Previously, you could be unsatisfied with the stock lens, but it’s not an issue anymore – because the HQ camera has no stock lens. The Raspberry Pi chooses not to include a lens but have curiously endorsed a little-known reseller – CGL.

In multiphoton excitation microscopy, fluorescent dyes are excited by absorbing the energy of two or more photons simultaneously (see Figure 1). For two-photon excitation, The dyes are excited at around twice the wavelength used in ordinary fluorescence observations, since the photon energy is inversely proportional to the wavelength. Because the excitation probability of the fluorescent dye in  two-photon excitation is proportional to the square of excitation light intensity, only the area proximal to the focal point, where the photon density is high, can be excited; tomographic images can then be obtained by scanning the laser beam (Figure 2, in which the non-excited area is grayed). The multiphoton excitation laser scanning microscopy provides similar tomographic images to the confocal LSM through the different principles.

Brian Herman and Victoria E. Centonze Frohlich - Department of Cellular and Structural Biology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78229.

Thomas J. Fellers and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.