Imagining the future of optical microscopy: everything, everywhere, all at once

Abstract

The optical microscope has revolutionized biology since at least the 17^th Century. Since then, it has progressed from a largely observational tool to a powerful bioanalytical platform. However, realizing its full potential to study live specimens is hindered by a daunting array of technical challenges. Here, we delve into the current state of live imaging to explore the barriers that must be overcome and the possibilities that lie ahead. We venture to envision a future where we can visualize and study everything, everywhere, all at once - from the intricate inner workings of a single cell to the dynamic interplay across entire organisms, and a world where scientists could access the necessary microscopy technologies anywhere.

Fig. 1. Example of an event-driven microscopy experiment.

a Scheme of an event-triggered STED (etSTED) experiment. Images from widefield calcium imaging of Oregon Green 488 bAPTA-1 in neurons (blue, top left images) are analyzed by a real-time analysis pipeline (light gray, bottom images). Detection of an event (small green box) triggers modality-switching to STED imaging at the corresponding location (red, top right image stack). b Schematic diagram of the etSTED microscope set-up, combining widefield and STED imaging under a control widget. Images are reproduced with permission from ref. .

Fig. 2. Examples of currently available tools that, if integrated with other methods, could potentially pave the way towards the goal of imaging “everything”.

a Target lipids that are transphosphatidylated by phospholipase D (PLD) can be visualized using click chemistry. The bottom images show labeled sites of PLD activity in HeLa cells. Scale bar = 10 µm (bottom left image), 50 µm (bottom right image). b Unmixed spectral image of an artificial mixture of 120 differently labeled E. coli. Scale bar = 25 µm. c Representative stimulated Raman scattering images of five melanoma cell lines (showing decreased differentiation from left to right) corresponding to the lipid peak (2,845 cm⁻¹; top row in red), protein peak (2940 cm⁻¹; middle row in blue), and the ratio of lipid/protein (bottom row). Scale bar = 20 µm. The images in (a–c) are reproduced with permission from refs. ^,,, respectively.

Fig. 3. Examples of different available technologies to image biological samples with greater depth and/or larger FOV.

a Comparison of different corrections, including the use of adaptive optics, on images of a live human stem cell-derived organoid expressing dynamin and clathrin obtained using a lattice light sheet microscope. b The top image shows a schematic of a MiniScope equipped with a GRIN lens mounted on a mouse’s head. The bottom image shows a representative fluorescent image of medium spiny neurons labeled with GCaMP6s. The traces indicate calcium transients from ROIs 1–9. Scale bar = 100 µm. c Projected image of the entire volume of a female Drosophila obtained using a Mesolens. Scale bar = 1 mm. d Benzyl alcohol/benzyl benzoate (BABB)-cleared Xenopus tropicalis tadpole stained for Atp1a1 (Alexa Fluor 594, orange) and nuclei (DAPI, grayscale). The sample was first imaged on a mesoSPIM light sheet microscope, and then using a Schmidt objective. The large Schmidt FOV allows imaging of both the entire head (~800 μm across) and individual developing photoreceptors in the eye. Scale bar = 500 µm, 100 µm, 50 µm, and 10 µm, respectively, for images going left to right. e Photoacoustic images of a 3-D reconstructed maximum amplitude projection and corresponding color-encoded depth-resolved image of a volunteer’s right hand. The blood vessel network can be clearly visualized. Scale bar = 3 cm. The images in (a–e) are reproduced with permission from refs. ^,,,,, respectively.

Fig. 4. A vision of the future of live imaging.

A culmination of various tool developments, integrated with easy and equitable access to these technologies, will enable imaging anytime, anything and anywhere, thereby powering biological breakthroughs.