As diaphanoscopic light source an SMD-LED XMLDCL-00-R250-00C5AAA02 LED (Cree, Durham, USA), including a red, green, blue and white LED (RGBW-LED) was used with peak wavelengths of 640 nm, 520 nm, 450 nm and 445 nm (for the blue peak of the white LED). The white LED had a CCT of 6000 K. The dimensions of the LED was 5 mm × 5 mm and it was mounted on a copper board, which was connected to a copper bar to dissipate the heat from the LEDs. For further heat reduction a 33.5 mm long light pipe PLP5-1000 (Bivar, Irvine (CA), USA) was placed close to the LEDs. The tip of this light pipe was applied for transscleral illumination of the fundus and was thus in contact with the sclera. Light pipe, LED board, copper bar and power cables were positioned using a 3D-printed framework. The design of these components are new compared to the existing RGBW-LED pen in [16], resulting in much better heat dissipation and therefore higher maximum LED intensities. The aluminum housing of the pen was 25 mm in diameter. Since too much heat is harmful to the sclera, the temperature was measured at the tip of the light pipe. The thermometer employed was the UT320 (UNI-T, Dongguan City, China). The control unit and the handling of the LED pen was similar to [16]. One improvement is the new minicomputer to control the LED intensities, Pi 4 Model B minicomputer (Raspberry Foundation, Cambridge, Great Britain). The current applied at each LED could be controlled independently in 5% steps with “ + ” and “ − “ buttons. 100% corresponds to a current of 350 mA. The spectral irradiance \(}\uplambda }\) emitted by the RGBW-LED pen was measured with the calibrated spectroradiometer CAS 140D, which was connected to an integrating sphere (ISP 250 UV, Instrument Systems, Munich, Germany), illustrated in Fig. 1a. The aperture of the integrating sphere was 3 mm in diameter, representing the contact surface of the diaphanoscopic pen on the eye.

a Set-up for determining the emitted irradiance of the advanced RGBW-LED pen. Light was captured by an integrating sphere and detected with a spectroradiometer. b Set-up for tests with the advanced RGBW-LED pen on ex-vivo porcine eyes. The RGBW-LED pen illuminated the eye transsclerally. With an attachment lens and a camera the fundus of the eye could be displayed. c Set-up for transmission measurement of sclera and choroidea. A section of the sclera and choroidea was illuminated with a fiber and transmitted light was collected with an integrating sphere and detected by a spectrometer. d Set-up for determining the CCT the ophthalmologist perceives during surgery. The eye was illuminated transsclerally by the advanced RGBW-LED pen and light in front of the pupil was detected by a spectroradiometer. (Color figure online)

For the test of the RGBW-LED pen, porcine eyes from a local butcher were used. Measurements were performed on the day of enucleation. The eyes were stored in balanced salt solution at 4 °C in the fridge and were divided in two groups, eyes with brown/dark iris and eyes with blue/light iris. As the iris color is correlated to the amount of melanin in the choroid-retinal pigment epithelial cells [22], the eyes were called strong and less pigmented in our study. The transscleral illumination tests on porcine eyes were performed with single LEDs or LEDs in combination. If a LED was turned on the intensity was set to 100% (corresponding to 350 mA LED current). The pen was placed at the posterior pole of the eye and light was transmitted through the eyewall into the interior space. This light was detected and sharp images were generated with a camera (D5600, Nikon, Tokio, Japan), in front of the porcine lens (Fig. 1b). In addition, a biconvex attachment lens was placed on the cornea so that the retina could be brought into focus. The camera exposure time, HDR setting as well as aperture were kept constant when photographing the eye. Only the ISO value of the camera was adjusted to the light conditions of the transscleral illumination.

With the wavelength dependent irradiance of the LED pen, \(}\uplambda }\) , the photochemical and thermal hazard to the retina could be calculated. According to the international standard DIN EN ISO 15004-2:2007 [17] and Sliney et al. (2005) [18] the photochemical hazard was calculated by weighting the irradiance \(}\uplambda }\) with the photochemical hazard weighting function A(λ), see Eq. (1). EA−R is called the “with A(λ) weighted irradiance on the retina”. The photochemical hazard weighting function is high in the low wavelength region, therefore the sum in Eq. (1) had to be calculated between 305 and 700 nm. For determining the thermal retinal hazard, the irradiance was weighted with the thermal hazard weighting function R(λ), see Eq. (2). EVIR-R is called the “with R(λ) weighted visible and infrared irradiance on the retina”. As the thermal hazard is the highest in the visible and infrared region, the sum in Eq. (2) ranges from 380 to 1400 nm. The standard for ophthalmic instruments EN ISO 15004-2:2007 specifies limit values for these photochemical and thermal weighted irradiances. The limit values for the photochemical hazard is 0.22 mW/cm2 and for the thermal hazard 700 mW/cm2 [17, 18]. No retinal damage is to be expected below these values. If the photochemical limit of 0.22 mW/cm2 was exceeded, the maximum exposition time had to be considered and was calculated as in Eq. (3).

$$}_} - }}} = \mathop \sum \limits_}}}^}}} }\lambda \cdot }\lambda \cdot \Delta \lambda$$

(1)

$$}_} - \;}}} = \mathop \sum \limits_}}}^}}} }\lambda \cdot }\lambda \cdot \Delta \lambda$$

(2)

$$}_}} = \frac}/}}}}_} - }}} }}$$

(3)

Since light is attenuated by the sclera and choroidea before it hits the retina, the photochemical and thermal hazard were also reduced. To determine the actual exposure on the retina, the irradiance \(}\uplambda }\) was weighted by the transmission \(}\uplambda }\) of the sclera and choroidea. Equations (1–3) then were replaced by Eqs. (4–6).

$$}_} - }\;}}} \mathop \sum \limits_}}}^}}} }\lambda \cdot }\lambda \cdot }\lambda \cdot \Delta \lambda$$

(4)

$$}_} - }\;}}} \mathop \sum \limits_}}}^}}} }\lambda \cdot }\lambda \cdot }\lambda \cdot \Delta \lambda$$

(5)

$$}_}} = \frac}/}}}}_} - }}} }}$$

(6)

The transmission of the sclera and choroidea was determined as illustrated in Fig. 1c. Light from a xenon lamp LQX 1000 SC (Linos, Goettingen, Germany) and a halogen lamp SLS201 L/M (Thorlabs, Newton, USA) were combined with a bifurcation fiber BFY400MS02 (Thorlabs, Newton, USA) and coupled into an ophthalmological illumination fiber TotalView Endoillumination Probe including illuminated scleral depressor 3269.B06 (D.O.R.C., Zuidland, The Netherlands). The fiber tip was in contact with the tissue and light, which was transmitted through the tissue, was captured by the integrating sphere MSP REFLTRANS1 (Mountain Photonics GmbH, Landsberg am Lech, Germany) and detected with a spectrometer AvaSpec-HSC 1024 × 58TEC-EVO (Avantes, Apeldoorn, The Netherlands), which was connected to the integrating sphere via another fiber M114L01 (Thorlabs, Newton, USA).

To determine the CCT the surgeon perceives during surgery for various settings of intensities for different LED combinations a spectroradiometer BTS256-LED (Gigahertz-Optik GmbH, Türkenfeld, Germany) with an aperture of 10 mm diameter was placed in front of the eye. The inside of the eye was illuminated transsclerally with the RGBW-LED illumination pen with various combinations and intensities of the LEDs. A schematic sketch is illustrated in Fig. 1d). The CCT was provided by the software of the spectroradiometer.