A near-total internal reflection fluorescence microscope comprising: a stage comprising an object region configured to support a sample; an illumination subsystem comprising a prism disposed beneath the object region and a light source operable to transmit light onto the prism, causing the prism to illuminate the sample; and an imaging subsystem comprising an objective lens unit disposed beneath the prism and an image capture device operable to receive an image passed through the objective lens unit from the object region; wherein the microscope further comprises: an activation subsystem comprising an activation light source configured to transmit activation light that is spectrally distinct from the illumination light onto the sample; and a calcium imaging subsystem to provide excitation light for a light-sensitive calcium-indicating protein, wherein the illumination subsystem, the imaging subsystem, the activation subsystem, and the calcium imaging subsystem are disposed beneath the object region, the object region supports the sample by means of a sample dish with a transparent bottom portion, wherein the sample dish can hold an aqueous medium and provide access to the sample from above, and the light source transmits light into a side of the prism to cause the prism to illuminate a region of the sample at the bottom portion of the sample dish approximately 10 microns thick without exhibiting total internal reflection. a stage comprising an object region configured to support a sample; an illumination subsystem comprising a prism disposed beneath the object region and a light source operable to transmit light onto the prism, causing the prism to illuminate the sample; and an imaging subsystem comprising an objective lens unit disposed beneath the prism and an image capture device operable to receive an image passed through the objective lens unit from the object region; wherein the microscope further comprises: an activation subsystem comprising an activation light source configured to transmit activation light that is spectrally distinct from the illumination light onto the sample; and a calcium imaging subsystem to provide excitation light for a light-sensitive calcium-indicating protein, wherein the illumination subsystem, the imaging subsystem, the activation subsystem, and the calcium imaging subsystem are disposed beneath the object region, the object region supports the sample by means of a sample dish with a transparent bottom portion, wherein the sample dish can hold an aqueous medium and provide access to the sample from above, and the light source transmits light into a side of the prism to cause the prism to illuminate a region of the sample at the bottom portion of the sample dish approximately 10 microns thick without exhibiting total internal reflection. an activation subsystem comprising an activation light source configured to transmit activation light that is spectrally distinct from the illumination light onto the sample; and a calcium imaging subsystem to provide excitation light for a light-sensitive calcium-indicating protein, wherein the illumination subsystem, the imaging subsystem, the activation subsystem, and the calcium imaging subsystem are disposed beneath the object region, the object region supports the sample by means of a sample dish with a transparent bottom portion, wherein the sample dish can hold an aqueous medium and provide access to the sample from above, and the light source transmits light into a side of the prism to cause the prism to illuminate a region of the sample at the bottom portion of the sample dish approximately 10 microns thick without exhibiting total internal reflection.