Electrospray has been used as one of the most powerful techniques for the analysis of biological samples. Recently, we have developed the probe electrospray (PESI) (modified version of the conventional electrospray) that uses the fine solid needle. In this method, the probe needle moves up and down along the vertical axis by a motor-driven system. At the highest position of the probe needle, electrospray is generated by applying a high voltage. In this study, we used PESI directly to the biological samples such as human milk, bovine milk, beverages, mouse brains, flower petals, plant leaves, bananas, tulip bulbs, etc. Strong ion signals for almost all samples were obtained. The amount of liquid sample picked up by the needle is as small as a few pL or less, making the PESI one of the most promising less-invasive analysis and imaging for biological samples. PESI may be useful as versatile and ready-to-use semi-on line analyses in the fields of agriculture, food science, medicine, pharmaceutical, etc. An example of imaging for mouse brain is demonstrated.